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Satellite DNA-containing gigantic introns in a unique gene expression program during Drosophila spermatogenesis
Jaclyn M Fingerhut, Jessica V. Moran, View ORCID ProfileYukiko M Yamashita
Jaclyn M Fingerhut
Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI 48109, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
Jessica V. Moran
Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA
Yukiko M Yamashita
Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI 48109, USA Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109, USA
ORCID record for Yukiko M Yamashita
For correspondence: yukikomy@umich.edu
Intron gigantism, where genes contain megabase-sized introns, is observed across species, yet little is known about its purpose or regulation. Here we identify a unique gene expression program utilized for the proper expression of genes with intron gigantism. We find that two Drosophila genes with intron gigantism, kl-3 and kl-5, are transcribed in a spatiotemporal manner over the course of spermatocyte differentiation, which spans ~90 hours. The introns of these genes contain megabases of simple satellite DNA repeats that comprise over 99% of the gene loci, and these satellite-DNA containing introns are transcribed. We identify two RNA-binding proteins that specifically localize to kl-3 and kl-5 transcripts and are needed for the successful transcription or processing of these genes. We propose that genes with intron gigantism require a unique gene expression program, which may serve as a platform to regulate gene expression during cellular differentiation.
Introns, non-coding elements of eukaryotic genes, often contain important regulatory sequences and allow for the production of diverse proteins from a single gene, adding critical regulatory layers to gene expression (Shaul, 2017). Curiously, some genes contain introns so large that more than 99% of the gene locus is non-coding. In humans, neuronal and muscle genes are enriched amongst those with the largest introns (Scherer, 2010). One of the best-studied large genes, Dystrophin, a causative gene for Duchenne Muscular Dystrophy, spans 2.2Mb, only 11kb of which is coding. A large portion of the remaining non-coding sequence is comprised of repetitive DNA-rich introns (Pozzoli et al., 2002). While intron size ('gigantism') is conserved between mouse and human, there is little sequence conservation within the introns, implying the functionality of intron gigantism (Pozzoli et al., 2003).
The Drosophila Y chromosome provides an excellent model for studying intron gigantism. Approximately 80% of the 40Mb Y chromosome is comprised of repetitive sequences, primarily satellite DNAs, which are short tandem repeats, such as (AATAT)n (Figure 1A)(Carvalho, 2002; Hoskins et al., 2002; Lohe et al., 1993; Peacock et al., 1978). The Drosophila Y chromosome encodes fewer than 20 genes (Carvalho et al., 2015), six of which are collectively known as the 'fertility factors' (Brosseau, 1960; Gatti, 1983; Hazelrigg, 1982; Kennison, 1981). One of the fertility factors, kl-3, which encodes an axonemal dynein heavy chain (Carvalho et al., 2000; Goldstein et al., 1982; Hardy et al., 1981), spans at least 4.3Mb (Bonaccorsi et al., 1988; Gatti, 1983; Pimpinelli, 1986), while its coding sequence is only ~14kb (Figure 1A). This is due to the large satellite DNA rich-introns, some of which are megabases in size that comprise more than 99% of the kl-3 locus. The other fertility factors (kl-1, kl-2, kl-5, ks-1, ks-2), have a similar gene structure, possessing large introns of repetitive satellite DNAs (Gatti, 1983). These six large Y chromosome genes are solely expressed during spermatogenesis (Bridges, 1916; Hardy et al., 1981; Marsh and Wieschaus, 1978).
Figure 1. The Y-loop gene kl-3 is expressed in a spatiotemporal manner during SC development.
(A) Diagram of the Drosophila Y chromosome. Regions enriched for satellite DNA (checkered pattern), locations of the fertility factor genes (magenta) and the Y-loop forming regions (black bars) with associated satellite DNA sequences are indicated. Enlarged is a diagram of the Y-loop B gene kl-3. Exons (vertical rectangles), introns (black line), intronic satellite DNA repeats (dashed line) and regions of kl-3 targeted by RNA FISH probes (colored bars). (B) Diagram of Drosophila spermatogenesis: GSCs (attached to the hub) produce mitotically-amplifying SGs, which become SCs. SCs develop over an 80–90 hour G2 phase before initiating the meiotic divisions. (C) Top: SC nucleus model showing the Y-loops in the nucleoplasm. DNA (white), Y chromosome (green), Y-loops A and C (red) and Y-loop B (blue). Bottom: RNA FISH for the Y-loop gene intronic transcripts in a SC nucleus. Y-loops are visualized using probes for Y-loops A and C (Cy3-(AAGAC)6, red) and Y-loop B (Cy5-(AATAT)6, blue). DAPI (white), SC nucleus (yellow dashed line) and nuclei of neighboring cells (white dashed line). Bar: 10μm. (D-H) RNA FISH to visualize kl-3 expression in wildtype testes. Exon 1 (blue), kl-3 intron (Alexa488-(AATAT)6, green), Exon 14 (red) and DAPI (white). (D) Apical third of the testis through the end of SC development (yellow dashed line). Bar: 75μm. (E-H) Single SC nuclei (yellow dashed line) at each stage of kl-3 expression. Nuclei of neighboring cells (white dashed line) and cytoplasmic mRNA granules (yellow arrows). Bar: 10μm. Inset: kl-3 mRNA granule (yellow dashed line). (I) RNA FISH against kl-3 following kl-3 RNAi (bam-gal4>UAS-kl-3TRiP.HMC03546). Single late SC nucleus (yellow dashed line) and nuclei of neighboring cells (white dashed line). Bar: 10μm.
In the Drosophila testis, germ cells undergoing differentiation are arranged in a spatiotemporal manner, where the germline stem cells (GSCs) reside at the very apical tip and differentiating cells are gradually displaced distally (Figure 1B) (Yamashita, 2018). GSC division gives rise to spermatogonia (SG), which undergo four mitotic divisions with incomplete cytokinesis to become a cyst of 16 SGs. 16-cell SG cysts enter meiotic S phase, at which point they become known as spermatocytes (SCs). SCs have an extended G2 phase, spanning 80–90 hours, prior to initiation of the meiotic divisions. During this G2 phase, the cells increase approximately 25 times in volume and the homologous chromosomes pair and segregate into individual chromosome territories (Figure 1C) (Fuller, 1993; McKee et al., 2012). During this period, SCs transcribe the majority of genes whose protein products will be needed for meiotic division and spermiogenesis (Gould-Somero and Holland, 1974; Olivieri and Olivieri, 1965; Schafer et al., 1995). Gene expression in SCs is thus tightly regulated to allow for timely expression of meiotic and spermiogenesis genes (White-Cooper and Caporilli, 2013).
It has long been known that three of the Y-chromosome-associated genes that contain gigantic introns (kl-5, kl-3 and ks-1, Figure 1A) form lampbrush-like nucleoplasmic structures in SCs, named Y-loops [denoted as loops A (kl-5), B (kl-3), and C (ks-1), (Figure 1C, D)] (Bonaccorsi et al., 1988). Y-loop structures reflect the robust transcription of underlying genes, and have been observed across Drosophilids, including D. simulans, D. yakuba, D. pseudoobscura, D. hydei and D. littoralis (Hess, 1967; Piergentili, 2007). Much of the fundamental knowledge about Y-loops comes from D. hydei, which forms large, cytologically distinct Y-loops (Hackstein et al., 1982), leading to the discovery that these structures are formed by the transcription of large loci comprised of repetitive DNAs (Hochstenbach et al., 1994; Huijser et al., 1990; Trapitz et al., 1992; Vogt, 1986a; Vogt, 1986b; Wlaschek et al., 1988). Interestingly, in D. pseudoobscura, which contains a 'neo-Y' (not homologous to the ancestral Y chromosome), Y-loops are thought to be formed by Y-linked genes instead of by the kl-3, kl-5 and ks-1 homologs, which are on the autosomes (Chang and Larracuente, 2017), suggesting that Y-loop formation is a unique characteristic of Y-linked genes, instead of being a gene-specific phenomenon.
The transcription/processing of such gigantic genes/RNA transcripts, in which exons are separated by megabase-sized introns, must pose a significant challenge for cells. However, how genes with intron gigantism are expressed and whether intron gigantism plays any regulatory role in gene expression remain largely unknown. In this study, we began addressing these questions by using the Y-loop genes as a model, and describe the unusual nature of the gene expression program associated with intron gigantism. We find that transcription of Y-loop genes progresses in a strictly spatiotemporal manner, encompassing the entire ~90 hours of SC development: the initiation of transcription occurs in early SCs, followed by the robust transcription of the satellite DNA from the introns, with cytoplasmic mRNA becoming detected only in late SCs. We identify two RNA-binding proteins, Blanks and Hephaestus (Heph), which specifically localize to the Y-loops, and show that they are required for robust transcription and/or proper processing of the Y-loop gene transcripts. Mutation of the blanks or heph genes leads to sterility due to the loss of Y-loop gene products. Our study demonstrates that genes with intron gigantism require specialized RNA-binding proteins for proper expression. We propose that such unique processing may be utilized as an additional regulatory mechanism to control gene expression during differentiation.
Transcription of a Y-loop gene, kl-3, is spatiotemporally organized
To start to investigate how the expression of Y-loop genes may be regulated, we sought to monitor their expression during SC development. In previous studies using D. hydei, when two differentially-labeled probes against two intronic repeats of the Y-loop gene DhDhc7(y) (homologous to D. melanogaster kl-5) were used for RNA fluorescent in situ hybridization (FISH), expression of the earlier repeat preceded that of the later repeat (Kurek et al., 1996; Reugels et al., 2000), leading to the idea that Y-loop genes might be transcribed as single, multi-megabase, transcripts. Consistently, Miller spreading of SC chromosome with transcripts still bound to DNA showed the long Y-loop transcripts (de Loos et al., 1984; Grond, 1983). However, transcription of exons was not visualized and extensive secondary structures were present in the Miller spreads, leaving it unclear whether the entire gene region is transcribed as a single transcript.
By using differentially-labeled probe sets designed for RNA FISH to visualize 1) the first exon, 2) the satellite DNA (AATAT)n repeats found in multiple introns including the first (Bonaccorsi and Lohe, 1991; Lohe et al., 1993), and 3) exon 14 (of 16) of kl-3 (Figure 1A, Supplementary file 1), we found that kl-3 transcription is organized in a spatiotemporal manner: transcript from the first exon becomes detectable in early SCs, followed by the expression of the (AATAT)n satellite from the introns, then finally by the transcript from exon 14 in more mature SCs (Figure 1D). These results suggest that transcription of kl-3 takes the entirety of SC development, spanning ~90 hours. The pattern of transcription is consistent with the model proposed for Y-loop gene expression in D. hydei: the gene is likely transcribed as a single transcript that contains the exons and gigantic introns, although we cannot exclude the possibility of other mechanisms, such as the trans-splicing of multiple individually transcribed exons.
Based on the expression pattern of early exon, (AATAT)n satellite-containing introns, and late exon, SC development can be subdivided into four distinct stages (Figure 1E-H). In stage 1, only exon 1 transcript is apparent (Figure 1E). In stage 2, the expression of intron transcript is detectable, while the signal from the first exon remains strong (Figure 1F). Stage 3 is defined by the addition of late exon signal, indicating that transcription is nearly complete (Figure 1G). Stage 4 is characterized by the presence of exon probe signals in granule-like structures in the cytoplasm (Figure 1H), which likely reflect kl-3 mRNA localizing to ribonucleoprotein (RNP) granules, as they never contain intron probe signal. These granules are absent following RNAi-mediated knockdown of kl-3 (bam-gal4>UAS-kl-3TRip.HMC03546, Figure 1I), confirming that they reflect kl-3 mRNA. The same pattern of expression was seen for the Y-loop gene kl-5 (see below, Figure 4B, C), suggesting that transcription of the other Y-loop genes proceeds in a similar manner.
Together, these results show that the gigantic Y-loop genes, including megabases of intronic satellite DNA repeats, are transcribed in a process that spans the entirety of SC development, culminating in the formation of mRNA granules in the cytoplasm near the end of the 80–90 hour meiotic G2 phase. This pattern of Y-loop gene transcription indicates that they are under precise regulation.
Identification of genes that may regulate the transcription of the Y-loop genes
Considering the size of the Y-loop gene loci and their satellite DNA-rich introns, transcription of Y-loop genes likely utilizes unique regulatory mechanisms. To start to understand such a genetic program, we performed a screen (See Methods and Supplementary file 2). Briefly, a list of candidates was curated using a combination of gene ontology (GO) terms, expression analysis, predicted functionality and reagent availability, resulting in a final list of 67 candidate genes. Candidates were screened for several criteria including protein localization, fertility, and Y-loop gene expression. Among these, two genes, blanks and hephaestus (heph), exhibit localization patterns and phenotypes that reveal critical aspects of Y-loop gene regulation and were further studied. Several proteins, including Boule (Cheng et al., 1998), Hrb98DE (Lowe et al., 2014), Pasilla (Lowe et al., 2014; Redhouse et al., 2011) and Rb97D (Heatwole and Haynes, 1996), were previously shown to localize to the Y-loops but displayed no detectable phenotypes in Y-loop gene expression in SCs using RNAi-mediated knockdown and/or available mutants (Supplementary file 2), and were not further pursued in this study.
Blanks and Heph are RNA binding proteins that specifically localize to the Y-loops and are required for fertility
Blanks, a RNA binding protein with multiple dsRNA binding domains, is primarily expressed in SCs in the testis. Blanks has been shown to be important for post-meiotic sperm development and male fertility (Gerbasi et al., 2011; Sanders and Smith, 2011). Blanks', ability to bind RNA was found to be necessary for fertility (Sanders and Smith, 2011). In order to assess Blanks' localization within the SC nucleus, testes expressing GFP-Blanks were processed for RNA FISH with probes against the intronic satellite DNA transcripts [(AATAT)n for Y-loop B/kl-3, (AAGAC)n for Y-loops A/kl-5 and C/ks-1 (Bonaccorsi et al., 1990)]. We found that GFP-Blanks exhibits strong localization to Y-loop B (Figure 2A).
Figure 2. Blanks and Heph localize to the Y-loops and are required for fertility.
(A, B) RNA FISH against the Y-loop gene intronic transcripts in flies expressing GFP-Blanks (A) or Heph-GFP (B). Y-loops A and C (Cy3-(AAGAC)6, red), Y-loop B (Cy5-(AATAT)6, blue), GFP (green), SC nucleus (yellow dashed line) and nuclei of neighboring cells (white dashed line). Bar: 10μm. (C) Table listing Y-loop designation, associated gene, the satellite DNA repeats found within the introns and whether the Y-loop is bound by Heph or Blanks. (D-I) Phase contrast images of seminal vesicles in blanks controls (C), blanksKG00084/Df (D), heph controls (E), heph2/Df (F), bam-gal4>UAS-kl-3TRiP.HMC03546 (G) and bam-gal4>UAS-kl-5TRip.HMC03747 (H). Sperm within the seminal vesicle (red arrowhead) and extruded sperm (red arrow). Bar: 100μm. (J) Schematic of IC progression during individualization. Nucleus (blue), axoneme (black), ICs (red) and cytoplasm (green). (K-V) Phalloidin staining of early ICs (K-P) and late ICs (Q-V) in indicated genotypes. Phalloidin (actin, red) and DAPI (white). Bar : 10μm.
Heph, a heterogeneous nuclear ribonucleoprotein (hnRNP) homologous to mammalian polypyrimidine track binding protein (PTB), is a RNA-binding protein with multiple RNA recognition motifs (RRMs) that is expressed in the testis (Davis et al., 2002). Heph has also been implicated in post-meiotic sperm development and male fertility (Robida et al., 2010; Robida and Singh, 2003). By using a Heph-GFP protein trap (p(PTT-GC)hephCC00664) combined with RNA FISH to visualize the Y-loop gene intronic transcripts, we found that Heph-GFP localizes to Y-loops A and C (Figure 2B). It should be noted that the heph locus encodes 25 isoforms and the Heph-GFP protein trap likely represents only a subset of heph gene products. A summary of Y-loop designation, gene, intronic satellite DNA repeat, and binding protein is provided in Figure 2C.
We confirmed previous reports that blanks is required for male fertility (Gerbasi et al., 2011; Sanders and Smith, 2011). By examining the seminal vesicles for the presence of motile sperm, we found that seminal vesicles from control siblings contain abundant motile sperm (Figure 2D, 13% empty, 87% normal, n=46) while seminal vesicles from blanks mutants (blanksKG00084/Df(3L)BSC371) lack motile sperm (Figure 2E, 96% = empty, 4% greatly reduced, n=58). We also confirmed previous reports that heph is required for fertility (Castrillon et al., 1993; Robida et al., 2010). Seminal vesicles from heph mutants (heph2/Df(3R)BSC687) also lack motile sperm (Figure 2G, 100% empty, n=21), while those from control siblings contain motile sperm (Figure 2F, 5% empty, 95% normal, n=57).
Previous studies (Gerbasi et al., 2011; Robida et al., 2010; Sanders and Smith, 2011) reported that blanks and heph mutants are defective in sperm individualization, one of the final steps in sperm maturation, where 64 interconnected spermatids are separated by individualization complexes (ICs) that form around the sperm nuclei and migrate in unison along the sperm tails, removing excess cytoplasm and encompassing each cell with its own plasma membrane (Figure 2J) (Fabian and Brill, 2012). When the F-actin cones of IC were visualized by Phalloidin staining, it became clear that ICs form properly in all genotypes (Figure 2K-N), but become disorganized in the late ICs in blanks and heph mutants, preventing completion of individualization (Figure 2Q-T).
The sterility and individualization defects observed in blanks and heph mutants are reminiscent of the phenotypes observed in flies lacking axonemal dynein genes including kl-5 and kl-3, the Y-loop A and B genes (Carvalho et al., 2000; Fatima, 2011; Gepner and Hays, 1993; Goldstein et al., 1982; Hardy et al., 1981; Timakov and Zhang, 2000). Upon RNAi mediated knockdown of kl-3 and kl-5 (bam-gal4>UAS-kl-3TRiP.HMC03546 or bam-gal4>UAS-kl-5TRiR.HMC3747), motile sperm are not found in the seminal vesicles (Figure 2H, I, kl-3: 100% empty, n=81, kl-5: 94% empty, 6% reduced, n=50) and a scattering of late ICs is observed (Figure 2O-P, U-V). Based on these observations, we hypothesized that the sterility and ICs defects observed in blanks and heph mutants may arise due to failure in the expression of the Y-loop genes.
blanks is required for transcription of the Y-loop B gene kl-3
As blanks was found to localize to Y-loop B, we first determined whether there were any overt defects in Y-loop B formation or kl-3 expression in blanks mutants. To this end, we performed RNA FISH to visualize the Y-loop gene intronic transcripts in blanks mutants. Compared to control testes where intronic satellite DNA transcripts from all Y-loops become detectable fairly early in SC development and quickly reach full intensity (Figure 3A), the signal from the Y-loop B intronic transcripts remains faint in blanks mutants (Figure 3B). The expression of Y-loops A and C is comparable between control and blanks mutant testes (Figure 3A, B).
Figure 3 blanks is required for kl-3 expression.
(A, B) RNA FISH against the Y-loop gene intronic transcripts in blanks control (A) and blanksKG000804/Df (B). Testis outline (yellow dashed line), Y-loops A and C (Cy3-(AAGAC)6, red), Y-loop B (Cy5-(AATAT)6, blue) and DAPI (white). Comparable stage SCs are indicated by yellow arrows. Bar: 50μm. High magnification images of single SCs at a comparable stage are provided below. SC Nuclei (yellow dashed line) and nuclei of neighboring cells (white dashed line). Bar: 10μm. (C-F) RNA FISH against kl-3 in blanks control (C, D) and blanksKG00084/Df (E, F). Exon 1 (blue), kl-3 intron (Alexa488-(AATAT)6, green), Exon 14 (red) and DAPI (white). (C, E) Apical third of the testis through the end of SC development (yellow dashed line). Bar: 75μm. (D, F) Single late SC nucleus (yellow dashed line). Nuclei of neighboring cells (white dashed line) and mRNA granules (yellow arrows). Bar: 10μm. (G) Western blot for Kl-3–3X FLAG in the indicated genotypes. (H) RT-qPCR in blanksKG00084/Df for kl-3 using the indicated primer sets. Primer locations are designated by red bars on the gene diagram. Data was normalized to GAPDH and sibling controls. Mean ±SD (p-value ***≤0.001 t-test between mutant and control siblings, exact p-values listed in Source Data 1)
In addition to a reduction in the expression of the intronic satellite DNA repeats of Y-loop B/kl-3, expression of kl-3 exons is also reduced in blanks mutants. By performing RNA FISH using exonic and intronic (AATAT)n probes for Y-loop B/kl-3, we found that blanks mutant flies display an overall reduction in signal intensity for both intronic satellite repeats and exons compared to controls (Figure 3C, E). Moreover, cytoplasmic kl-3 mRNA granules are rarely detected in blanks mutants (Figure 3F). The same results are obtained following RNAi mediated knockdown of blanks (bam-gal4>UAS-blanksTRiP.HMS00078, Supplementary Figure 1). These results suggest that blanks is required for robust and proper expression of Y-loop B/kl-3 and for the production of kl-3 mRNA granules, likely at the transcriptional level. Consistently, we found that the amount of Kl-3 protein is greatly diminished in blanks mutants, confirming that blanks is required for proper expression of Y-loop B/kl-3 (Figure 3G).
To obtain a more quantitative measure of kl-3 expression levels in control and blanks mutant testes, we performed RT-qPCR. Primers were designed to amplify early (close to the 5' end), middle, and late (close to the 3' end) regions of kl-3. For each region, two sets of primers were designed: one primer set spanned a satellite-DNA-containing large intron and another spanned a normal size intron (Figure 3H, bars denote spanned intron, and Supplementary file 3). All primer sets show a detectable drop in kl-3 mRNA levels in blanks mutants (Figure 3H). We noted a detectable drop between the early primer sets (~75% reduction in expression levels compared to controls) and the middle/late primer sets (~95% reduction in expression levels compared to controls), raising the possibility that blanks mutants may have difficulty transcribing this Y-loop gene soon after encountering the first satellite DNA containing gigantic intron. In summary, the RNA binding protein Blanks localizes to Y-loop B and allows for the robust transcription of the Y-loop B gene kl-3.
In contrast to Y-loop B/kl-3 expression, Y-loop A/kl-5 expression appeared normal in blanks mutants. We designed RNA FISH probes against kl-5 in the same manner as for kl-3 (i.e. early exon, intron and late exon) (Figure 4A, Supplementary file 1). We found that transcription of Y-loop A/kl-5 follows a spatiotemporal pattern similar to that of Y-loop B/kl-3 (Figure 4B, C): early exon transcripts becomes detectable in early SCs while kl-5 mRNA granules are not detected in the cytoplasm until near the end of SC development (Figure 4B, C). No overt differences are observed in kl-5 expression in blanks mutants and kl-5 mRNA granules are observed in the cytoplasm (Figure 4D, E). By RT-qPCR with primers for kl-5 designed similarly as described above for kl-3 (Figure 3H), we found a mild reduction in kl-5 expression in blanks mutants (Figure 4F). However, considering the fact that the kl-5 mRNA granule is correctly formed in blanks mutant testes (Figure 4E), this reduction may not be biologically significant. The mild reduction in kl-5 transcript in blanks mutants could be an indirect effect caused by defective Y-loop B expression. Alternatively, it is possible that a small amount of (AATAT)n satellite, which is predicted to be present in the last intron of kl-5 (Bonaccorsi and Lohe, 1991; Hoskins et al., 2015), might cause this mild reduction in kl-5 expression in blanks mutant testes.
Figure 4 blanks is not required for kl-5 expression.
(A) Diagram of the Y-loop A gene kl-5. Exons (vertical rectangles), introns (black line) and intronic satellite DNA repeats (dashed line). Regions of kl-5 targeted by RNA FISH probes are indicated by the colored bars. (C-F) RNA FISH against kl-5 in blanks controls (C, D) and blanksKG00084/Df (E, F). Exons 1–6 (red), kl-5 intron (Cy5-(AAGAC)6, blue), Exons 16–17 (green, arrowhead indicates nuclear signal), DAPI (white). (C, E) Apical third of the testis through the end of SC development (yellow dashed line). Bar: 75μm. (D, F) Single late SC nucleus (yellow dashed line). Nuclei of neighboring cells (white dashed line) and mRNA granules (yellow arrows). Bar: 10μm. (G) RT-qPCR in blanksKG00084/Df for kl-5 using the indicated primer sets. Primer locations are designated by red bars on the gene diagram. Data was normalized to GAPDH and sibling controls. Mean ±SD (p-value ***≤0.001, t-test between mutant and control siblings, exact p-values listed in Source Data 2).
Blanks is unlikely to be a part of the general meiotic transcription program
It is well known that SCs utilize a specialized transcription program in order to transcribe the vast majority of genes required for meiosis and spermiogenesis (Lin et al., 1996; White-Cooper and Caporilli, 2013; White-Cooper et al., 1998). This program is executed by two groups of transcription factors: tMAC and the tTAFs. The tMAC (testis-specific meiotic arrest complex) complex has both activating and repressing activities and has been shown to physically interact with the core transcription initiation machinery (Ayyar, 2003; Beall et al., 2007; Doggett et al., 2011; Jiang, 2003; Jiang et al., 2007; Lu and Fuller, 2015; Perezgasga et al., 2004). The tTAFs (testis-specific TATA binding protein associated factors) are homologs of core transcription initiation factors (Chen et al., 2005; Hiller et al., 2004; Hiller et al., 2001; Laktionov et al., 2014). tMAC and the tTAFs function cooperatively to regulate meiotic gene expression. To examine whether blanks is part of this established meiotic transcription program, we examined the expression of fzo and Dic61B, known targets of the SC-specific transcriptional program (Laktionov et al., 2014; White-Cooper et al., 1998), which are located on autosomes and do not have gigantic introns (Supplementary file 1). In contrast to mutants for the tMAC component aly (aly2/5P), which has drastically reduced levels of fzo and Dic61B transcripts, the expression of these genes is not visibly affected in blanks mutants (Supplementary Figure 2), suggesting that blanks is not a part of the SC-specific transcriptional program involving tTAF and tMAC. Instead, blanks is likely uniquely involved in the expression of the Y-loop genes.
Heph is required for processing transcripts of the Y-loop A gene kl-5
As Heph-GFP localized to Y-loops A and C, we first examined whether Y-loops A and C displayed any overt expression defects in heph mutants as seen in blanks mutants (Figure 3B). When we performed RNA FISH to visualize the Y-loop gene intronic transcripts in heph mutants, the overall expression levels of both (AAGAC)n and (AATAT)n satellites appear unchanged between control and heph mutant testes (Figure 5A, B). However, we noted that the morphology of Y-loops A and C is altered in heph mutants, adopting a less organized, diffuse appearance (Figure 5B), whereas all Y-loops in control SCs show characteristic thread-like or globular morphologies (Figure 5A). Y-loop B appears unchanged between controls and heph mutants (Figure 5A, B). These results indicate that heph may be important for structurally organizing Y-loop A and C transcripts, without affecting overall transcript levels.
Figure 5 heph is required for kl-5 mRNA production.
(A, B) RNA FISH against the Y-loop gene intronic transcripts in heph controls (A) and heph2/Df (B). Single late SC nuclei (yellow dashed line), nuclei of neighboring cells (white dashed line), Y-loops A and C (Cy3-(AAGAC)6, red), Y-loop B (Cy5-(AATAT)6, blue) and DAPI (white). Bar: 10μm. (C-F) RNA FISH against kl-5 in heph controls (C, D) and heph2/Df (E, F). Exons l-6 (red), kl-5 intron (Cy5-(AAGAC)6, blue), Exons 16–17 (green, arrowhead indicates nuclear signal) and DAPI (white). (C, E) Apical third of the testis through the end of SC development (yellow dashed line). The bulbous shape of heph2/Df is a known phenotype that can occur with this allele (Castrillon et al., l993). Bar: 75μm. (D, F) Single late SC nuclei (yellow dashed line). Nuclei of neighboring cells (white dashed line) and mRNA granules (yellow arrows). Bar: 10μm. (G) RT-qPCR in heph2/Df for kl-5 using the indicated primer sets. Primer locations are designated by red bars on the gene diagrams. Data was normalized to GAPDH and sibling controls. Mean ±SD (p-value **≤0.01, ***≤0.001, t-test between mutant and control siblings, exact p-values listed in Source Data 3).
We next examined expression pattern of kl-5 exons together with the Y-loop A/C intronic satellite [(AAGAC)n] as described in Figure 4. Overall expression levels of kl-5 appear to be unaltered in heph mutant testes (Figure 5C, E). However, in contrast to control testes (Figure 5D), heph mutant testes rarely have cytoplasmic kl-5 mRNA granules in late SCs (Figure 5E), suggesting that heph mutants affect kl-5 mRNA production without affecting transcription in the nucleus. heph mutants may be defective in processing the long repetitive regions of transcripts to generate mRNA (e.g. splicing, mRNA export). Similar to blanks mutants, heph mutants do not affect the expression of fzo or Dic61B (Supplementary Figure 2), indicating that heph is not a member of the more general meiotic transcription program. Instead, heph, like blanks, appears to specifically affect the expression of Y-loop A/kl-5 to which it localizes.
RT-qPCR showed that heph mutants only exhibit a moderate reduction in kl-5 expression (Figure 5G), which is in accordance with the RNA FISH results described above. A similar moderate reduction in kl-5 mRNA is observed in blanks mutants (Figure 4F), which do not affect kl-5 mRNA granule formation. Thus, it is unlikely that the reduction in kl-5 expression levels alone causes the lack of kl-5 mRNA granules in heph mutant SCs. Instead, we postulate that mRNA granule formation is dependent on proper processing of primary transcripts, which may be defective in heph mutants.
Surprisingly, we found that kl-3 mRNA granules are also absent in heph mutants, although Y-loop B/kl-3 expression levels in the nucleus appear to be unaffected (Figure 6A-D). RT-qPCR showed a similar moderate reduction in kl-3 mRNA in heph mutants (Figure 6E) as was observed in kl-5 mRNA (Figure 5G). Consistent with the absence of cytoplasmic kl-3 mRNA granules, Kl-3 protein levels are dramatically reduced in heph mutant testes (Figure 6F). This is unexpected as Heph protein does not localize to Y-loop B (Figure 2B) or affect Y-loop B morphology (Figure 5A, B). It is possible that some of the predicted 25 isoforms of Heph are not visualized by Heph-GFP, and these un-visualized isoforms might localize to and regulate Y-loop B/kl-3 expression. Alternatively, this may be an indirect effect of defective Y-loop A and C expression and/or structure.
Figure 6 kl-3 expression is affected in heph mutants
(A - D) RNA FISH against kl-3 in heph controls (A, B) and heph2/Df (C, D). Exon 1 (blue), kl-3 intron (Alexa488-(AATAT)6, green), Exon 14 (red) and DAPI (white). (A, C) Apical third of the testis through the end of SC development (yellow dashed line). Bar: 75μm. (B, D) Single late SC nuclei (yellow dashed line). Nuclei of neighboring cells (white dashed line) and mRNA granules (yellow arrows), Bar: 10μm. (E) RT-qPCR in heph2/Df for kl-3 using the indicated primer sets. Primer locations are designated by red bars on the gene diagrams. Data was normalized to GAPDH and sibling controls. Mean ±SD (p-value **≤0.01 ***≤0.001, t-test between mutant and control siblings, exact p-values listed in Source Data 4). (F) Western blot for Kl-3-3X FLAG in the indicated genotypes. (G) Model for the Y-loop gene transcriptional program.
Taken together, our results show that Blanks and Heph, two RNA binding proteins, are essential for the expression of Y-loop genes, but are not members of the more general meiotic transcription program. As Y-loop genes are essential for sperm motility and fertility, the sterility observed in blanks and heph mutants likely stems from defects in Y-loop gene expression. Blanks and Heph highlight two distinct steps (transcriptional processivity and RNA processing) in a unique Y-loop gene expression program.
The existence of the Y chromosome lampbrush-like loops of Drosophila has been known for the last five decades (Hess and Meyer, 1963; Meyer et al., 1961), however little is known as to how Y-loop formation and expression is regulated and whether these SC-specific structures are important for spermatogenesis. Here, we identified a Y-loop gene-specific expression program that functions in parallel to the general meiotic transcriptional program to aid in the expression and processing of the gigantic Y-loop genes. Our results suggest that genes with intron gigantism, such as the Y-loop genes and potentially other large genes such as Dystrophin, require specialized mechanisms for proper expression.
The phenotypes of blanks and heph, the two genes identified to be involved in this novel expression program, highlight two distinct steps of the Y-loop gene specific expression program (Figure 6G). Blanks was originally identified as an siRNA binding protein, but no defects in small RNA mediated silencing were observed in the testes of blanks mutants (Gerbasi et al., 2011; Sanders and Smith, 2011). We found that blanks is required for transcription of Y-loop B/kl-3, as nuclear transcript levels were visibly reduced in blanks mutants, leading to the lack of both kl-3 mRNA granules in the cytoplasm and Kl-3 protein. As Blanks' ability to bind RNA was previously found to be required for male fertility (Sanders and Smith, 2011), we speculate that Blanks may bind to newly synthesized nascent kl-3 RNA, which contain megabases of satellite DNA transcripts, such that transcripts' secondary/tertiary structures do not interfere with transcription (Zhang and Landick, 2016). It is possible that RNA polymerases, which have inherently low efficiency (Darzacq et al., 2007), might require Blanks to increase their processivity, allowing them to transcribe through repetitive DNA sequences (Fitz et al., 2018).
Heph has been implicated in a number of steps in RNA processing and translational regulation (Kafasla et al., 2012; Sawicka et al., 2008; Valcarcel and Gebauer, 1997; Wagner and Garcia-Blanco, 2001), but Heph's exact role in the testis remained unclear despite its requirement for male fertility (Robida et al., 2010; Robida and Singh, 2003). We found that heph mutants fail to generate kl-5 cytoplasmic mRNA granules even though nuclear transcript levels appeared minimally affected. This suggests that heph may be required for processing the long repetitive transcripts. For example, heph might be required to ensure proper splicing of the Y-loop gene pre-mRNAs, which is predicted to be challenging as the splicing of adjacent exons becomes exponentially more difficult as intron length increases (Shepard et al., 2009). Y-loop genes may utilize proteins like Heph to combat this challenge.
These results highlight the presence of a unique program tailored toward expressing genes with intron gigantism. Although the functional relevance of intron gigantism remains obscure, our results may provide hints as to the possible functions of intron gigantism. Even if intron gigantism did not arise to serve a specific function, once it emerges, the unique gene expression program that can handle intron gigantism must evolve to tolerate the burden of gigantic introns, as indicated by our study on blanks and heph mutants. Ultimately, the presence of a unique gene expression program for genes with gigantic introns would provide a unique opportunity to regulate gene expression. Once such systems evolve, other or new genes may start utilizing such a gene expression program to add an additional layer of complexity to the regulation of gene expression. For example, in the case of Y-loop genes, the extended time period required for the transcription of the gigantic Y-loop genes (~80-90 hours) might function as a 'developmental timer' for SC differentiation. Similar to this idea, it was shown that the expression of two homologous genes, knirps (kni) and knirps-like (knrl), is regulated by intron size during embryogenesis in Drosophila. Although knrl can perform the same function as kni in embryos, mRNA of knrl is not produced due to the presence of a relatively large (14.9kb) intron (as opposed to the small (<1kb) introns of kni), which prevents completion of knrl transcription during the short cell cycles of early development (Rothe et al., 1992). Thus, intron size can play a critical role in the regulation of gene expression. Alternatively, satellite DNA-containing gigantic introns could act in a manner similar to enhancers, recruiting transcriptional machinery to the Y-loop genes to facilitate expression (Shaul, 2017).
In summary, our study provides the first glimpse at how the expression of genes with intron gigantism requires a unique gene expression program, which acts on both transcription and post-transcriptional processing.
Fly Husbandry
All fly stocks were raised on standard Bloomington medium at 25°C, and young flies (1- to 3-day-old adults) were used for all experiments. Flies used for wild-type experiments were the standard lab wild-type strain yw (y1w1). The following fly stocks were used: heph2 (BDSC:635), Df(3R)BSC687 (BDSC: 26539), blanksKG00084 (BDSC:13914), Df(3L)BSC371 (BDSC:24395), p(PTT-GC)hephCC00664 (BDSC:51540), UAS-kl-3TRiP.HMC03546 (BDSC:53317), UAS-blanksTRiP.HMS00078 (BDSC:33667), UAS-kl-5TRiP.HMC03747 (BDSC:55609), and C(1)RM/C(1;Y)6, y1w1f1/0 (BDSC:9460) were obtained from the Bloomington Stock Center (BDSC). GFP-blanks (GFP-tagged blanks expressed by endogenous promoter) was a gift of Dean Smith (Sanders and Smith, 2011). bam-gal4 was a gift of Dennis McKearin (Chen and McKearin, 2003). The aly2 and aly5P stocks were a gift of Minx Fuller (Lin et al., 1996).
The Y chromosome in the heph deficiency strain Df(3R)BSC687 appeared to have accumulated mutations that resulted in abnormal Y-loop morphology. This Y chromosome was replaced with the yw Y chromosome for all experiments described in this study.
The kl-3-FLAG strain was constructed by Fungene (fgbiotech.com) using CRISPR mediated knock-in of a 3X-FLAG tag at the C-terminus of kl-3 using homology-directed repair.
RNA Fluorescent in situ Hybridization
All solutions used for RNA FISH were RNase free. Testes from 2–3 day old flies were dissected in 1X PBS and fixed in 4% formaldehyde in 1X PBS for 30 minutes. Then testes were washed briefly in PBS and permeabilized in 70% ethanol overnight at 4°C. Testes were briefly rinsed with wash buffer (2X saline-sodium citrate (SSC), 10% formamide) and then hybridized overnight at 37°C in hybridization buffer (2X SSC, 10% dextran sulfate (sigma, D8906), 1mg/mL E. coli tRNA (sigma, R8759), 2mM Vanadyl Ribonucleoside complex (NEB S142), 0.5% BSA (Ambion, AM2618), 10% formamide). Following hybridization, samples were washed three times in wash buffer for 20 minutes each at 37°C and mounted in VECTASHIELD with DAPI (Vector Labs). Images were acquired using an upright Leica TCS SP8 confocal microscope with a 63X oil immersion objective lens (NA = 1.4) and processed using Adobe Photoshop and ImageJ software.
Fluorescently labeled probes were added to the hybridization buffer to a final concentration of 50nM (for satellite DNA transcript targeted probes) or 100nM (for exon targeted probes). Probes against the satellite DNA transcripts were from Integrated DNA Technologies. Probes against kl-3, kl-5, fzo, and Dic61B exons were designed using the Stellaris® RNA FISH Probe Designer (Biosearch Technologies, Inc.) available online at www.biosearchtech.com/stellarisdesigner. Each set of custom Stellaris® RNA FISH probes was labeled with Quasar 670, Quasar 570 or Fluorescein-C3 (Supplemental file 1).
For strains expressing GFP (e.g. GFP-Blanks, Heph-GFP), the overnight permeabilization in 70% ethanol was omitted.
RT-qPCR
Total RNA from testes (50 pairs/sample) was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. 1μg of total RNA was reverse transcribed using SuperScript III® Reverse Transcriptase (Invitrogen) followed by qPCR using Power SYBR Green reagent (Applied Biosystems). Primers for qPCR were designed to amplify only mRNA. For average introns, one primer of the pair was designed to span the two adjacent exons.
Primers spanning large introns could only produce a PCR product if the intron has been spliced out. Relative expression levels were normalized to GAPDH and control siblings. All reactions were done in technical triplicates with at least two biological replicates. Graphical representation was inclusive of all replicates and p-values were calculated using a t-test performed on untransformed average ddct values. Primers used are listed in Supplementary file 3.
Testes (40 pairs/sample) were dissected in Schneider's media at room temperature within 30 minutes, the media was removed and the samples were frozen at −80°C until use. After thawing, testes were then lysed in 200uL of 2X Laemmli Sample Buffer + βME (BioRad, 161–0737). Samples were separated on a NuPAGE Tris-Acetate gel (3–8%, 1.5mm, Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore) using NuPAGE transfer buffer (Invitrogen) without added methanol. Membranes were blocked in 1X TBST (0.1% Tween-20) containing 5% nonfat milk, followed by incubation with primary antibodies diluted in 1X TBST containing 5% nonfat milk. Membranes were washed with 1X TBST, followed by incubation with secondary antibodies diluted in 1X TBST containing 5% nonfat milk. After washing with 1X TBST, detection was performed using the Pierce® ECL Western Blotting Substrate enhanced chemiluminescence system (Thermo Scientific). Primary antibodies used were anti–α-tubulin (1:2,000; mouse, monoclonal, clone DM1a; Sigma-Aldrich) and anti-FLAG (1:2,500; mouse, monoclonal, M2, Sigma-Aldrich). The secondary antibody was horseradish peroxidase (HRP) conjugated anti-mouse IgG (1:10,000; Jackson ImmunoResearch Laboratories).
Screen for the identification of proteins involved in Y-loop gene expression
Initially, ~2200 candidate genes were selected based on gene ontology (GO) terms (e.g.. "mRNA binding", "regulation of translation", "spermatid development"). These genes were cross-referenced against publicly available RNAseq data sets (i.e.: FlyAtlas, modENCODE) and only those genes predicted to be expressed in the testis were selected. Additionally, candidate genes were eliminated if they are known to be involved in ubiquitous processes (e.g. general transcription factors, ribosomal subunits) or processes that are seemingly unrelated to those associated with the Y-loop genes (e.g. mitochondrial proteins, GSC/SG differentiation, mitotic spindle assembly). Finally, candidates were limited to those with available reagents for localization and/or phenotypic analysis, leaving a final list of 67 candidate genes (Supplementary file 2). If available, we first analyzed protein localization for each candidate. If candidate proteins did not localize to SCs or the Y-loops, they were not further examined. If the candidate was found to be expressed in SCs or if no localization reagents were available, then RNAi mediated knockdown or mutants were used to examine Y-loop gene expression for any deviations from the expression pattern described in Figure 1D-H and to assess fertility. As Y-loop genes are all essential for sperm maturation (Hardy et al., 1981), any genes essential for Y-loop gene expression should also be needed for fertility. All selection criteria and a summary of phenotypes observed can be found in Supplementary file 2.
Phalloidin Staining
Testes were dissected in 1X PBS, transferred to 4% formaldehyde in 1X PBS and fixed for 30 minutes. Testes were then washed in 1X PBST (PBS containing 0.1% Triton-X) for at least 60 minutes followed by incubation with Phalloidin-Alexa546 (ThermoFisher, a22283, 1:200) antibody in 3% bovine serum albumin (BSA) in 1X PBST at 4°C overnight. Samples were washed for 60 minutes in 1X PBST and mounted in VECTASHIELD with DAPI (Vector Labs). Images were acquired using an upright Leica TCS SP8 confocal microscope with a 63X oil immersion objective lens (NA = 1.4) and processed using Adobe Photoshop and ImageJ software.
Seminal vesicles were dissected in 1X PBS and transferred to slides for live observation by phase contrast on a Leica DM5000B microscope with a 40X objective (NA = 0.75) and imaged with a QImaging Retiga 2000R Fast 1394 Mono Cooled camera. Images were adjusted in Adobe Photoshop.
Supplementary Figure 1 blanks RNAi recapitulates the phenotypes observed in blanks mutants
(A) RNA FISH against the Y-loop gene intronic transcripts in bam-gal4>UAS-blanksTRiP.HMS00078 testes. Testis outline (yellow dashed line), Y-loops A and C (Cy3-(AAGAC)6, red), Y-loop B (Cy5-(AATAT)6, blue) and DAPI (white). Comparable stage SC (yellow arrow, compare to Figure 3A-B). Bar: 50 μm. High magnification image of a single SC at a comparable stage (compare to Figure 3A, B) is provided below. SC nucleus (yellow dashed line) and nuclei of neighboring cells (white dashed line). Bar: 10μm. (B, C) RNA FISH against kl-3 in bam-gal4>UAS-blanksTRiP.HMS00078 testes. Exon 1 (blue), kl-3 intron (Alexa488-(AATAT)6, green), Exon 14 (red) and DAPI (white). (B) Apical third of the testis through the end of SC development (yellow dashed line). Bar: 75μm. (C) Single late SC nucleus (yellow dashed line). Nuclei of neighboring cells (white dashed line) and mRNA granules (yellow arrows), Bar: 10μm.
Supplementary Figure 2 blanks and heph are not part of the meiotic transcriptional program
RNA FISH against fzo (A-E) and Dic61B (A'-E') in blanks controls (A), blanksKG00084/Df (B), heph controls (C), heph2/Df (D), and aly2/5P (E). Apical third of the testis through the end of SC development (yellow dashed line). DAPI (white). Bar: 75μm..
Supplementary Tables
Supplementary file 1: RNA FISH probes
Supplementary file 2: Screen
Supplementary file 3: qPCR primers
We thank Dean Smith, Dennis McKearin, Minx Fuller, the Bloomington Stock Center and the Developmental Studies Hybridoma Bank for reagents. We thank the Yamashita laboratory and Drs. Sue Hammoud and Lei Lei for discussions and comments on the manuscript, Dr. Ruth Lehmann for helpful suggestions and Dr. Jiandie Lin for sharing equipment. This work was supported by the Howard Hughes Medical Institute (YMY) and the NIH Cellular and Molecular Biology Training Grant T32-GM007315 (JMF).
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Robida, M., V. Sridharan, S. Morgan, T. Rao, and R. Singh. 2010. Drosophila polypyrimidine tract-binding protein is necessary for spermatid individualization. Proc Natl Acad Sci U S A. 107: 12570–12575.
Robida, M.D., and R. Singh. 2003. Drosophila polypyrimidine-tract binding protein (PTB) functions specifically in the male germline. EMBO J. 22: 2924–2933.
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Posted December 11, 2018.
You are going to email the following Satellite DNA-containing gigantic introns in a unique gene expression program during Drosophila spermatogenesis
Jaclyn M Fingerhut, Jessica V. Moran, Yukiko M Yamashita


S/Y Drumbeat's helm after receiving the IMED treatment.
IMED has recently completed a successful helm station refit on S/Y Drumbeat. The 53-metre, Alloy Yachts-built ketch also received the IMED treatment in the form of reconfiguring shore power , seamless transfer and load sharing of generators all taken care of by the Westhaven-based company.
April and May will also see company representatives travel to West Palm Beach, Florida, where they'll install their custom-designed helm station for S/Y Genevieve, including two specially-commissioned carbon fibre wheels.
S/Y Drumbeat before IMED's refit work on her helm.
IMED has been successfully designing and building control stations since 2006, creating custom helm control panels from carbon fibre, membrane and glass. The company designs and builds in-house and prides itself on the research and development undertaken to achieve control panels that are watertight, user-friendly and aesthetically-pleasing.
Concept drawings of IMED's work on S/Y Genevieve's helm.
Based in ORAMS Marine, Auckland, IMED provides customised packages and solutions across electrical design and contracting, power generation solutions, shorepower conversion, alarms and monitoring systems and helm control stations.


3Dmoskcustoms is a small manufacturer from Italy, specialized in Alfa Romeo bodies, who do molds and bodies by handmade. All bodies have included 3D printet accessoires to build the body really scale.
I am the exclusive Dealer/Partner in Germany!
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text:
The Jara Festival was a great success in Dubai - Photo
Home page Gallery
Jara International Music Festival started in 2016, but for the first time this event was organized outside of the CIS.
Axar.az informs that, on May 3, at the Rixos Premium Dubai, one of the most luxurious hotels in Dubai, the brightest representatives of the Russian variety scene are Gregory Leps, EMIN, Leonid Agutin, Valeriya, Egor Krid, Jasmin, Alexander Panayotov, Ivanushki International, Artik & Asti, Vremya i Steklo, MARUV, Olga Buzova, Albina Canabayeva, Slava, Rodriguezzz, Rita Dakota and others.
A giant scene has been set up at the Jumeirah Beach Residence on the shores of the Persian Gulf. Within the framework of the festival, 3 tons of stage equipment, 300 kilowatt light and sound equipment were brought to Dubai. More than 3,000 spectators listened to a spectacular concert lasting over 5 hours outdoors. This was an unprecedented show in the Arab Emirates! "Jara" brought together Russian music lovers in Dubai and danced all the guests from the beach to the auditorium and danced with their favorite artists.
One of the organizers of the Jara festival, musician and businessman EMIN said: "Our Jara Festival is a promising project and one of the most exciting events. Therefore, I am very happy that the first place for her tour was Dubai, which meets all the advanced trends. On May 3, a very brilliant music show was performed by most popular artists. It's great to have so many of us in the evenings. Therefore, next year we will think about holding several festival days. "
"This is a great event for us, because Jara, the first time we've been to Dubai, is a great event," says Maxim Berin, president of BERIN IGLESIAS ART. None of the concerts held in Dubai before, can not be compared to the number of balls or total enthusiasm. The festival was so successful that a number of high-level concert halls in Dubai said they wanted to cooperate with us. We plan to turn into a traditional annual festival in Jamaica, which launches such a successful start in Dubai, the most talented, current and interesting artists of the Russian pop-scene, as well as their numerous fans.
"Rixos Premium Dubai" and "Jara" festival have given unforgettable rest and bright emotions to all the guests! Albina Canabayeva and Timur Rodriguez, as well as Yulia Baranovskaya and Konstantin Tarasyuk, have added holiday atmosphere and music to the concert.
The Jara Festival will be held in Baku on July 25-28, the coast of the Caspian Sea.
White phosphorus shells were found in Jabrayil - Photo
Mehriban Aliyeva`s last post in Shusha - Photo
The statues of these people returned to Shusha - Photo
President and the I Lady`s Shusha visit - Photo
Jeyhun Bayramov planted a tree in Pakistan - Photo
Pashinyan`s spokeswoman at the protest - Photo
Ilham Aliyev presented a sword - Photo
Pakistan gives Turkey's top diplomat high honour - Photo
Text of the statement signed by Aliyev-Putin-Pashinyan
Karabakh statement from Lavrov

redpajama_set_name: RedPajamaCommonCrawl


Money is a critical resource as demonstrated by every company that has gone through bankruptcy or has been on the verge of bankruptcy.
Generally speaking cash is also the ultimate constraint of the public sector, as the budget dictates the maximum value that the public-sector organization, a non-profit organization by definition, is able to draw. Limiting the working capital of an organization to the extent that lack of cash limits the throughput happens also to some profit organizations.
This article focuses on companies for which the cash limitation is a direct threat on their survival. This kind of cash constraint is unique because it is imperative to find the immediate way to go out of the situation.
People who are knowledgeable in TOC are aware that any specific constraint of the organization should dictate policies and norms of behavior that could be different than with another constraint. This dependency on the constraint is even more noted when the constraint lies with cash, which prevents purchasing the required materials and the use of capacity and by that disrupts the life line of the organization. The immediate result is that revenues are blocked. These revenues could have been used to generate more revenues and reduce the pressure on cash. In such a case money is both the goal and the absolutely required capacity to continue the business. This is a unique situation with very critical ramifications, which should lead the management to behave differently than in any other state. When lack of cash threatens the existence of the organization all the attention of the top management is consumed in fighting one payment crisis after another.
The seemingly complicating factor of money being the goal, the constraint and the direct threat to the organization makes the TOC insights regarding exploiting the constraint and subordinating everything else to it, especially strong. The good news are that the right behavior accelerates the regaining of cash in a non-linear way. The objective of this article is to point to ways to elevate the cash constraint. It is not a constraint a company can live with for too long. We believe that it is possible, many times, to get out of the cash constraint situation in 15-20 weeks.
The important generic insight for a survival cash-constraint situation is to understand the meaning of cash-to-cash cycle time and cash-velocity. This leads to being able to accelerate the cash-velocity and go out of the current state, even on the expense of the amount of revenues.
Cash to cash cycle time is the total time it takes from cash going out to cash coming in. In other words the time from the actual payment to a supplier until the client pays. We can measure the time by days or weeks as reasonable periods of time.
Cash Velocity (CV) is defined as the contribution-ratio of one unit of cash in one period of time. For any business every dollar invested in materials, or for providing the capacity required for a sale, is expected to yield, on average, more than one dollar. In other words, the ratio of the cash in to cash going-out should be higher than 1. The idea here is to get the ratio for one period of time, like a day or a week.
Suppose one dollar is the cost to buy materials and the finished item is sold for $2 dollars three weeks later. So, the going out cash is doubled in three weeks and should yield 4$ in another three weeks.
How much did the invested dollar yielded after just one week? The answer is NOT 33%, because if after one week we get $1.33, then we immediately invest the $1.33 to generate more sales and then after the second week we have 1.33*1.33 = $1.7689, and after the third week we should have: 1.33*1.33*1.33 = 2.35, not 2. In order to get the cash velocity (CV) of the situation where $1 would yield revenue after three weeks of $2 the CV = the 3rd root of 2 = 1.259, or 25.9% contribution rate in one week.
A company that is in the state of struggling with a cash constraint has operational lines working, but it needs to invest its limited cash to buy materials and then turn them into sales. These are the main body of the truly-variable-costs (TVC), the cost saved when one unit of output is not produced and sold. The quicker these dollars, used for TVC, are turned into Sales the amount of cash would grow until the state where the constraint moves to something else. The ratio of S/TVC, S stands for sales, representing the contribution of $1 invested in materials to cash coming from sales. This ratio is called: contribution-ratio.
The overhanging threat on the company is its ability to cover all the other operating expenses (OE). These are all the expenses the company has to carry to sustain the operational line alive. Without the OE there is no basic ability to survive. So, it is absolutely critical to have the amount of necessary OE in order to stay alive. The rest of the limited cash is to make more cash as fast as possible, until the company reaches the state where there is enough cash to support the full market demand. At that state the constraint might move to the market or to another resource.
The formal mathematical formula for cash velocity is: CV = ((S/TVC)^(1/n) -1).
The cash-to-cash cycle time is represented as n in the above definition.
Table 1 details the calculations for two different products P & Q.
It seems that given a choice, due to the lack of cash, between selling P and selling Q, that selling Q would generate cash faster, bringing the company to go out of the cash constraint situation earlier than focusing on selling P. This is not the intuitive answer, just to hint how far are most managers from the right answer when cash appears as a constraint.
Covering the critical operating expenses: the must-have expenses to keep going – OE.
Purchasing the absolutely required materials to enables sales. This is the TVC.
The minimal cash required for survival is n*OE, because whatever is purchased now will turn to revenues only in n periods. However, this amount does not leave any room for investing cash in order to get more cash. This means that once the company is left only with that amount of cash it is doomed. Question is what is the amount of cash that still provides a valid option to survive? We like to find out what cash leaves an option that after n periods the company would still have the same amount of cash. Let's call it adequate survival cash. So, we look for X cash that after n periods would yield exactly X cash. In the beginning we need first to put aside n*OE from the cash in order to cover the OE payments for all the periods, including the current one, until the new cash appears. The remaining cash of X-(n*OE) is used to purchase materials to sell end items after n periods getting X in revenues, allowing us to repeat the process. The invested cash in materials would yield c*(X-(n*OE)), where c is the contribution rate, S/TVC, and we like it to be equal to X.
Sufficient cash means the cash at hand is enough to cover the n*OE plus having enough to purchase whatever is needed to exploit the capacity and/or the maximum market demand. If the cost of materials that fully answer all the demand or the full capacity of the most loaded resource, then it is enough cash to be considered beyond the urgent need pull the company from its cash constraint situation.
Table 2 provides sample calculations for above parameters.
What the table shows is that having on hand cash between $4,001 and $4,500 and focusing on the Q product provides a much better way to bring the organization out of the cash constraint than by concentrating on the P product. If the market for the Q product is limited, and this is what constraining the company from investing more than $1,000 per period in purchasing materials, the organization has more cash than 4,500, and the internal constraint provides enough capacity also for the P product, then the organization can improve even more.
The above example is a simplified reality. Usually, while having on-hand only $2,000 there are already orders and materials in the pipeline. That means the cash-flow situation needs to be clearly specified week-by-week. But the principles are the same.
In cash constraint situation, the focus on generating as much cash and as fast as is possible through effective utilization of existing cash. A small increase or reduction in cash can make or break the organization. This unique property of cash impacting throughput non-linearly could help organizations to overcome cash constraint in a very short period of time. In most cases it may be possible to come out of cash constraint in less than three months by reducing cash to cash time, and by that accelerating the cash velocity.
Cash to cash cycle time (n) reduction has huge non-linear impact on throughput, cash availability, survival time, adequate cash requirement etc. Often just shrinking cash to cash cycle time is good enough to come out of the cash constraint situation provided the right measurements are in place.
The common TOC techniques of accelerating the flow of value to customers, through chocking the release of orders and the use of buffer management, are already good means to shorten the cash-to-cash cycle and increase cash-velocity.
Reducing the customer paying time. Giving the customer price reduction in exchange of significant faster payment is of paramount importance for achieving faster CV. It can be shown that even after providing 20% price discount to shrink customer payment time from 4 weeks to one week could exploit better the cash constraint. Of course, this might not be the right move when cash is not an active constraint.
Reducing the manufacturing lead-time is also of critical impact. While it is always good to reduce manufacturing lead-time, its impact on exploiting the cash constraint is even more critical. Even here, when possible, on top of all the known TOC techniques, to use overtime, for extra cash, to vastly reduce the lead-time and by that accelerate the revenues, then it has to be carefully checked.
This thinking on the special devastating impact of a cash constraint, and its practical meaning for exploiting the cash constraint, is a major contribution of the five focusing steps of TOC. Just remember the purpose here is to get rid of the cash constraint situation. Cash is not a resource to keep as a system constraint!
Hi Eli…thanks for the illuminating post.
The article gives very clear instructions on counter-intuitive decisions that need to be taken when faced with "Cash Constraint".
What should be decisions to avoid "Cash Constraint"? Take a case where the company has a market constraint, what should be the vendor payment policy vis-a-vis discount on purchases? What should be Cash Discount policy vis-a-vis receivables?
In India, the agro inputs market (fertilizers, seeds, pesticides etc) experience a seasonal demand coupled with long cash-to-cash cycles. The race to acquire as many customers as possible has given rise to practices of dumping physical inventory in the market and extending very generous credit policy to the channel partners. The smaller players in the market who do not have deep pockets get into the "Cash constraint" situation very often and then fold-up or have to scale down their activities after a period of frenetic growth. This growth is mostly on books as each year their receivables grow faster than the rate of growth of sales. This is accompanied with frenetic increase in SKUs being offered to the market and channel partners being added to the distribution ecosystem. The prevailing notion is 'in order to stay relevant', the company should expand and get market share. The only way to expand is by pumping in additional working capital.
I also think we should develop a detailed procedure for any organization is establishing and managing a money/cash/credit buffer. Ravi gives us the instructions for estimating the line where real damage is done by lack of enough cash. The buffer should protect the company from coming too close to that line. Some expediting cash procedure should be in place when the buffer penetrates the red. Very different means should be used when the company holds too much cash, which does not bring more cash, or bring too little of it.
I also agree with you about agriculture. Certainly the cash-to-cash cycle time is especially problematic. Another special perspective is dealing with very severe uncertainty, which is caused by the same causes for cash-constraint. I think I have the beginning of a decision-making support for agriculture, based on TOC, but accustomed to that different kind of environment.
Are you sure about REDUCING CASH VELOCITY? What is a reason of reducing cash velicity?
Dmitry, you are absolutely RIGHT. I meant increasing cash-velocity, or maybe shortening the cash-to-cash cycle.
One GOOD thing from such an error: it is evidence that people read and care enough to point when something is clearly wrong. I actually was already noted on the mistake by Mr. Kuroki from Japan. So, now I definitely should fix the post.


text:
by gliffen | November 10, 2009 | Uncategorized
Photo by Jeff Vanuga
THE LOOMING THREAT that the greater sage-grouse might be listed as an endangered species spurred Wyoming to adopt a new approach to protect the celebrated bird.
As a result, the Cowboy State has emerged as the regional leader in re-thinking sage-grouse policies, focusing its conservation efforts on sage-grouse "core" habitat areas.
Following Wyoming's lead, Montana's state game agency recently announced its own "core area" strategy.
There is some disagreement amongst Wyoming's conservation community about the value of this core area approach, said Sophie Osborn, wildlife biologist with the Wyoming Outdoor Council.
But Osborn said while the approach has some limitations, she believes Wyoming's attempt to conserve core sage-grouse habitat might be the West's best bet for maintaining viable sage-grouse populations, and for keeping the bird off the endangered species list.
Click here for a map of Wyoming's core sage-grouse habitat areas.
If properly implemented, Wyoming's approach could offer other states a model for finding a balance between energy development and the protection of this vulnerable species, Osborn said.
A MAJORITY OF THE NATION'S GREATER SAGE-GROUSE NOW LIVE IN WYOMING
Sage-grouse numbers have been in decline throughout the West for decades, and today 54 percent of the remaining greater sage-grouse in the United States live in Wyoming.
The U.S. Fish and Wildlife Service's decision whether to list the sage-grouse as an endangered species will be swayed, at least in part, by the strength and efficacy of Wyoming's policies toward the bird.
One criterion for the federal government's ultimate listing decision, Osborn said, is the answer to the following question. Are there regulatory mechanisms in place that will adequately protect the species?
"That's why Wyoming has worked so hard to develop this core sage-grouse concept," Osborn said. "To make sure there is an existing, adequate regulatory mechanism in place."
Wyoming's actions will likely play a key role in determining whether overall sage-grouse populations begin to stabilize or continue to decline.
"State and federal agencies, conservation groups, the energy industry—they all understand that what we do in Wyoming really matters," Osborn said. "It might seem as if we're obsessing over this bird, but Wyoming has to get this right, and we all know it."
In addition to the ecological motivations for preserving sage-grouse "core" areas, there are also economic incentives for keeping these grouse populations healthy.
If the sage-grouse were listed as an endangered species, energy extraction and other types of development could be subjected to more stringent regulations and possibly curtailed throughout the bird's range, and not only in core habitat areas. In a state that relies heavily on revenues from mineral extraction operations, such a possibility gets lawmakers' attention.
Sometimes just the threat of an endangered species listing can be the impetus for better wildlife management and development policies, Osborn said.
WIND DEVELOPERS LEFT OUT IN THE COLD?
The burgeoning wind energy industry has had a particularly difficult time adapting to Wyoming's sage-grouse core area conservation strategy.
As part of this strategy, Wyoming will not permit the development of wind farms in core areas until wind developers can show that their activities will not have a negative impact on sage-grouse populations.
In a letter sent to Secretary of the Interior Ken Salazar in July 2009, wind industry representatives warned that such a requirement would abruptly halt wind energy development in Wyoming's core areas and have "deleterious effects on renewable energy development across the Western United States."
The Wyoming governor's office maintains, however, that 86 percent of the state's "economically viable wind areas" are outside of sage-grouse core areas, and so the state still has ample opportunity to develop wind, without placing its, and the West's, sage-grouse populations at risk.
Some developers—frustrated by their inability to build wind farms in core areas until the effect of turbines on grouse is studied—feel that the wind industry was left out of the process when sage-grouse core areas were developed.
But Bob Budd, head of the Governor's sage-grouse task force, said wind energy development had not yet emerged as a prominent issue when the team developed the core area strategy. The push for widespread wind development is a relatively recent phenomenon in Wyoming.
A representative of the wind energy industry has since joined the Governor's sage-grouse team.
Horizon Wind Energy's Simpson Ridge Project near Medicine Bow was an early casualty of the state's rigorous stance on protecting core areas, even after the company committed to doing research to document the impacts of turbines on grouse.
However, bird advocates and energy watchdog groups, including the Wyoming Outdoor Council, argue that Horizon knew from the start about the important sage-grouse breeding areas near the company's proposed wind farm, and the company's promise to spearhead a research study was simply an attempt to justify sacrificing these areas.
"If, as we suspect, turbines cause grouse declines and displacement, we risk losing our healthiest grouse populations by conducting these studies in core areas," Osborn said. "Studies should be conducted in non-core areas, where we have fewer grouse to lose; otherwise, we increase the risk of an endangered species listing for the grouse."
Osborn's comments echo those of the U.S. Fish and Wildlife Service, which stated in July 2009 that building wind farms in core areas, even for research purposes, "would negate the usefulness of the core area concept," and undermine a key regulatory mechanism designed by Wyoming to protect its grouse populations.
Soon after the Fish and Wildlife Service issued its statement in support of making core areas off-limits to wind development, Horizon withdrew its construction and research proposal, because the project would have enveloped, and threatened, 15 active sage-grouse breeding grounds, or leks—where these chicken-like birds perform their distinctive courtship displays and choose mates.
Although the impacts of oil and gas development on sage-grouse are fairly well understood, scientists have yet to examine the effect of wind turbines on grouse. Until these impacts are known, agencies must rely on indirect, but relevant, scientific information that suggests that grouse will be displaced by wind turbines.
INTRODUCING TALL STRUCTURES INTO A SHORT ECOSYSTEM
Having evolved in open, treeless habitat—in what some refer to as the "sagebrush sea"—sage-grouse show a strong aversion to vertical structures. Tall objects do not occur in the bird's natural surroundings, and they can be used as perches by raptors that prey on grouse.
Sage-grouse populations have declined where trees, transmission lines, and oil and gas development have encroached on their habitat, Osborn said. In addition, grouse have been eliminated from habitats fragmented by roads and other forms of development.
Given these facts—coupled with the threat that sage-grouse could be listed as an endangered species without a robust state-level regulatory mechanism—the wind industry will have to focus for now on developing wind farms in areas with few or no sage-grouse, such as in the eastern portion of the state, Osborn said.
Many people in Wyoming and beyond view sage-grouse core area protection as the best hope for safeguarding a declining species, and the best chance to avoid an endangered species listing that would complicate not just wind energy, but all types of energy development in the West.
West Edge


The Rayo Lamp was manufactured by the Bradley & Hubbard Mfg. Co., in Meriden, CT. The flame spreader's first patent is Nov. 30, 1894, then called "Perfection" which were initially made under contract with the Standard Oil Co. One story is you got a free Rayo if you bought 15 gallons of Standard Oil kerosene. The Trademark for the name RAYO remained active under Eastern Standard, Esso, Humble Oil and lastly Exxon Corp. until 1988! B&H began to advertise and sell it to retailers like Montgomery Ward, (they must have paid royalties to Standard Oil for the use of the name). Its sole purpose was to help Standard Oil sell kerosene. Solid brass throughout and nickel plated. In 1911 they were $48.00 dozen with tripod. The Rayo was a round wick central draft lamp as were all the lamp makers, such as Pittsburgh Lamp, Plume & Atwood, Rochester, Edw. Miller, etc. – they were greatly out-performed by the new mantle Aladdins around 1907. These gave 60 watt from a much smaller wick and used half the fuel. The Rayo needs 30 minutes to an hour to REALLY warm up & produce good light, after that the flame can be turned up as high as 2 to 3" – (if you aren't concerned about breaking the chimney)! The Rayo is considered a fuel hog by today's collectors, which likely served the purposes of Standard Oil, its sponsor, quite well!
We carry a good selection of 10" white milk glass shades for Rayo and other similar lamps, also the tripod and rim shade holders, chimneys, wicks, and oil filler caps. We also restore, strip off the nickel that is left to get down to the solid brass and wire these old lamps for the public when desired. When the book on the Rayo lamp came out in 2001 it showed the Rayo with the cut off shade, rim and the tall chimney shown here.


Welcoming Center for New Pennsylvanians is a Philly non-profit that "connects newly arrived individuals from around the world with the economic opportunities."
Posted by Willy Franzen on July 20, 2013. Jobs updated daily.
Displaying 1-10 of 10,052 results.
When you're in the midst of a job search (especially when you're looking for your first job), it's easy to see immigration as a bad thing. You're already here, yet they (the government) are letting new people in to compete with you for those precious few entry level jobs. However, economic research shows that population growth drives economic growth, and immigration speeds that up considerably when you consider that a newborn baby typically takes more than 20 years to start contributing much in terms of productivity. I completely agree with Welcoming Center for New Pennsylvanians' belief that "a steady influx of new customers, workers, and entrepreneurs can reinvigorate Pennsylvania's aging population, renew its neighborhoods, and re-energize our economy." And that's why I think that the services that they offer are so important–they get new immigrants participating in the economy as quickly as possible. If you'd like to help make that happen, take a look at Welcoming Center for New Pennsylvanians' Jobs page. Right now they're hiring an Employment Specialist, and it looks like a great position for a new grad–even if employment isn't something that you know all that much about right now.
What do you think of Welcoming Center for New Pennsylvanians?


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Personal information is kept in secured locations and is only accessed by authorised personnel. Personal information kept electronically is handled with care and secured by user identifiers and passwords accessed only by authorised personnel.
Archived personal information is maintained online and is only accessed by authorised personnel.
The TNA only uses or discloses personal information about an individual for the primary purpose for which the information is collected. Personal information is not used or disclosed for a secondary (related) purpose, except in accordance with the National Privacy Principles (NPP's).
The TNA may use or disclose personal information for a secondary purpose where the secondary purpose is related to the primary purpose of collection (or, if the personal information is sensitive information, directly related to the primary purpose), and the individual would reasonably expect them to use or disclose the information for that secondary purpose.
The TNA may also use or disclose personal information about an individual for a secondary purpose if it has obtained the individual's consent (either express or implied).
The TNA will not otherwise disclose personal information unless the individual has given consent or that disclosure is required by law.
No personal information is disclosed to third parties for the purposes of the third parties direct marketing. Personal information (such as name and address), is used, from time to time, for the purpose of forwarding information in the form of journals, newsletters and circular letters, unless an individual requests that their personal information is not used for that purpose.
The TNA will, in a timely manner and at the written request of an individual, provide them with access to their personal information held by the TNA.
This Policy may be varied from time to time and an updated version will be posted on the TNA's website. Please check our website regularly to ensure that you have the most recent version of the Policy.