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In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.


Fecundity of vasPH165 females carrying different egfp-vas constructs, and expression levels from those constructs.(A) Fecundity. The y-axis indicates the number of eggs laid by individual females in the first three days after eclosion, and the x-axis identifies each egfp-vas construct that was tested. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vasPH165 (p<0.05). Error bars indicate the standard error of the mean (SEM), n (number of females tested) >50 for all genotypes. (B) Western blots (WB) comparing the expression level of eGFP-Vas in the ovaries from vas1/+ flies carrying the different constructs, using an anti-Vas antibody. α-Tubulin (α-Tub) serves as a loading control. egfp-vas+ was included in each blot for comparison. The eGFP-Vas bands migrate at different positions depending on the deletions that they carry. No Vasa protein was detected in the ovary lysate from vasPH165. The Vasa antibody raised against the full-length protein reacts with some mutant forms of Vasa such as vasΔ3-139 and vasΔ3-200 less strongly than with vas+ (see also supplementary material Fig. S1).
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f02: Fecundity of vasPH165 females carrying different egfp-vas constructs, and expression levels from those constructs.(A) Fecundity. The y-axis indicates the number of eggs laid by individual females in the first three days after eclosion, and the x-axis identifies each egfp-vas construct that was tested. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vasPH165 (p<0.05). Error bars indicate the standard error of the mean (SEM), n (number of females tested) >50 for all genotypes. (B) Western blots (WB) comparing the expression level of eGFP-Vas in the ovaries from vas1/+ flies carrying the different constructs, using an anti-Vas antibody. α-Tubulin (α-Tub) serves as a loading control. egfp-vas+ was included in each blot for comparison. The eGFP-Vas bands migrate at different positions depending on the deletions that they carry. No Vasa protein was detected in the ovary lysate from vasPH165. The Vasa antibody raised against the full-length protein reacts with some mutant forms of Vasa such as vasΔ3-139 and vasΔ3-200 less strongly than with vas+ (see also supplementary material Fig. S1).

Mentions: Females homozygous for vas deletions produce very few mature eggs (Styhler et al., 1998). To examine which domains of Vas are essential for oogenesis we expressed eGFP-Vas proteins with various mutations in a vasPH165 mutant background, and counted the number of eggs produced (Fig. 2A). For these and subsequent experiments, we selected individual transgenic lines that gave comparable to or higher levels of eGFP-Vas expression than the egfp-vas+ wild-type control (Fig. 2B; supplementary material Fig. S1).


In vivo mapping of the functional regions of the DEAD-box helicase Vasa.

Dehghani M, Lasko P - Biol Open (2015)

Fecundity of vasPH165 females carrying different egfp-vas constructs, and expression levels from those constructs.(A) Fecundity. The y-axis indicates the number of eggs laid by individual females in the first three days after eclosion, and the x-axis identifies each egfp-vas construct that was tested. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vasPH165 (p<0.05). Error bars indicate the standard error of the mean (SEM), n (number of females tested) >50 for all genotypes. (B) Western blots (WB) comparing the expression level of eGFP-Vas in the ovaries from vas1/+ flies carrying the different constructs, using an anti-Vas antibody. α-Tubulin (α-Tub) serves as a loading control. egfp-vas+ was included in each blot for comparison. The eGFP-Vas bands migrate at different positions depending on the deletions that they carry. No Vasa protein was detected in the ovary lysate from vasPH165. The Vasa antibody raised against the full-length protein reacts with some mutant forms of Vasa such as vasΔ3-139 and vasΔ3-200 less strongly than with vas+ (see also supplementary material Fig. S1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400588&req=5

f02: Fecundity of vasPH165 females carrying different egfp-vas constructs, and expression levels from those constructs.(A) Fecundity. The y-axis indicates the number of eggs laid by individual females in the first three days after eclosion, and the x-axis identifies each egfp-vas construct that was tested. Data from vasPH165 and vasPH165/Df(2L)A267 controls are presented at the far right. Asterisks indicate a significant increase compared to vasPH165 (p<0.05). Error bars indicate the standard error of the mean (SEM), n (number of females tested) >50 for all genotypes. (B) Western blots (WB) comparing the expression level of eGFP-Vas in the ovaries from vas1/+ flies carrying the different constructs, using an anti-Vas antibody. α-Tubulin (α-Tub) serves as a loading control. egfp-vas+ was included in each blot for comparison. The eGFP-Vas bands migrate at different positions depending on the deletions that they carry. No Vasa protein was detected in the ovary lysate from vasPH165. The Vasa antibody raised against the full-length protein reacts with some mutant forms of Vasa such as vasΔ3-139 and vasΔ3-200 less strongly than with vas+ (see also supplementary material Fig. S1).
Mentions: Females homozygous for vas deletions produce very few mature eggs (Styhler et al., 1998). To examine which domains of Vas are essential for oogenesis we expressed eGFP-Vas proteins with various mutations in a vasPH165 mutant background, and counted the number of eggs produced (Fig. 2A). For these and subsequent experiments, we selected individual transgenic lines that gave comparable to or higher levels of eGFP-Vas expression than the egfp-vas+ wild-type control (Fig. 2B; supplementary material Fig. S1).

Bottom Line: We identified novel domains in the N- and C-terminal regions of the protein that are essential for localization, transposon repression, posterior patterning, and pole cell specification.One such functional region, the most C-terminal seven amino acids, is specific to Vas orthologues and is thus critical to distinguishing Vas from other closely related DEAD-box helicases.Surprisingly, we also found that many eGFP-Vas proteins carrying mutations that would be expected to abrogate DEAD-box helicase function localized to the nuage and posterior pole, and retained the capacity to support oogenesis, although they did not function in embryonic patterning, pole cell specification, grk activation, or transposon repression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 3649 Promenade Sir William Osler, Montréal, QC H3G 0B1, Canada.

No MeSH data available.