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Mega Drain Or Synthesis Essay

Introduction

During early development, mitotic chromosome size scales to cell size; repeated cell division in the absence of embryo growth reduces cell size, and concomitantly, chromosome size decreases (Kieserman and Heald, 2011; Hara et al., 2013; Ladouceur et al., 2015). Chromosome size scaling is critical for life, as a single mitotic chromosome in an early embryo can be as large as the entire metaphase plate of smaller cells late in development (unpublished data). Previously, nuclear transport via the Ras-related nuclear protein (RAN) pathway was shown to play an important role in regulating mitotic chromosome size, implicating nuclear proteins in chromosome-size regulation (Hara et al., 2013; Ladouceur et al., 2015). To find proteins whose nuclear localization is critical for mitotic chromosome scaling, we devised and executed an RNAi screen of the Caenorhabditis elegans nucleome in a sensitized setting.

We performed our screen in a C. elegans strain that harbors an exceptionally long chromosome as the result of a telomere fusion event between the two longest chromosomes (5 and X; Lowden et al., 2008, 2011). This strain is viable in laboratory conditions and contains a genome size not significantly different from the wild-type parent strain. We predicted that depletions with a robust effect on chromosome length would lead to embryonic lethality in our long-chromosome strain but not in control animals. This strategy identified the centromere-specific histone H3 variant CENP-A (HCP-3/CPAR-1 in C. elegans, hereafter CENP-A) and topoisomerase-II (TOP-2 in C. elegans, hereafter topo-II), among 13 other proteins, as being required for viability in the presence of an exceptionally long chromosome (Fig. 1, a and b).

CENP-A–containing nucleosomes define centromeres and make up an estimated 2–5% of the metazoan genome. In C. elegans, as well as many other organisms, centromeres are not restricted to a single region of each chromosome, resulting in CENP-A dispersed along each chromosome (holocentric; Maddox et al., 2006; De Rop et al., 2012). Current hypotheses in both monocentric (regionalized) and holocentric organisms predict that patches of CENP-A chromatin assemble to form a unified centromeric chromatin region in mitosis (Blower et al., 2002). The mechanism of mitotic centromere assembly is unknown; however, CENP-A nucleosomes have increased internal rigidity that could potentially drive self-assembly on the outer face of mitotic chromosomes forming a linear array. In agreement with this hypothesis, in vitro reconstitution of synthetic arrays showed that CENP-A–containing nucleosomes are more condensed than bulk chromatin (Panchenko et al., 2011; Geiss et al., 2014). In C. elegans, CENP-A depletion results in each chromosome collapsing into a ball rather than forming rod-shaped chromosomes during mitosis, supporting the idea that CENP-A chromatin stretches are structurally important (Maddox et al., 2006). Here, we find, counterintuitively to our screen, that CENP-A chromatin levels positively correlate with chromosome length; lower levels of CENP-A lead to shorter chromosomes (see Results and discussion).

Topo-II is involved in numerous DNA metabolic processes through its enzymatic activity: DNA decatenation of loops (Nitiss, 2009). Topo-II localizes to the central long axis of monocentric mitotic chromosomes (Earnshaw et al., 1985; Gasser et al., 1986), and loss of topo-II function impairs chromosome assembly, leading to chromosome segregation defects in most eukaryotic cells (Uemura et al., 1987; Adachi et al., 1991; Hirano and Mitchison, 1993). In addition to mitotic errors, topo-II loss of function activates the G2 checkpoint and leads to decreased transcriptional activity (Downes et al., 1994; Mondal and Parvin, 2001). The C. elegans early embryo has weak checkpoint activity and is transcriptionally silent, allowing cell-cycle progression after topo-II depletion (Budirahardja and Gönczy, 2009). We find that topo-II levels are constant through early development, and it localizes spatially independent of CENP-A. Depletion of topo-II led to abnormally long chromosomes, consistent with a recent study in vertebrate cells (Farr et al., 2014).

In sum, our screen designed to identify proteins and required to limit chromosome length identified proteins that contribute to both shortening and lengthening of chromosomes. Our evidence supports the idea that CENP-A and topo-II localize and function independently to provide structure and to determine the length of holocentric mitotic chromosomes.

Results and discussion

A reverse genetic screen for proteins required to segregate an abnormally long chromosome

To identify proteins required for setting chromosome length, we individually RNAi depleted a subset (∼400) of proteins predicted to localize to the C. elegans nucleus in two conditions: a strain harboring a chromosomal fusion between 5 and X and the control parent strain (wild type; Lowden et al., 2008, 2011). Compared with wild-type C. elegans, our long-chromosome strain has the same genome size and indistinguishable viability in laboratory conditions (Lowden et al., 2008, 2011). To perform the screen, L4-stage larvae were incubated with individual clones from a library of bacteria expressing double-stranded RNAs (dsRNAs) targeting the nucleome (Fig. 1 a; Tursun et al., 2011). The viability of RNAi-treated progeny was scored 5 d later, and a range of phenotypes was recorded (Fig. 1 b). Our strategy identified 15 individually confirmed nuclear proteins as required for embryogenesis in our long-chromosome strain. The list includes chromatin modifiers (taf-1, trr-1, and swsn-3), chromosome architecture (his-2, top-2, cin-4, and cpar-1), and phosphorylation/ubiquitination (lin-41, plk-1, ubc-9, and air-2), as well as several with unknown function (taf-4, attf-6, mag-1, and pdcd-2). One of the 15 hits, Aurora-B (air-2), has been shown to be required for shortening an extra-long chromosome during budding yeast anaphase (Neurohr et al., 2011), revealing that our strategy identified proteins that impact chromosome size.

Figure 1.

RNAi screen of the C. elegans nucleome identifies chromosomal architecture proteins as regulators of chromosome size. (a) Diagrammatic flow and list of positive hits from the RNAi screen. Pink asterisks highlight potential regulators of chromosome length tested in this study. zts, zygotes. (b) Representative images of five different phenotypes and partial CENP-A RNAi observed after RNAi treatment of wild-type or long-chromosome strains.

Among our hits, two proteins previously reported as required for chromosome condensation and segregation in C. elegans stood out: CENP-A and topo-II (Fig. 1, a and b; Maddox et al., 2006; Bembenek et al., 2013). C. elegans uniquely expresses two orthologues of CENP-A; HCP-3 is the dominant form required for mitosis, whereas CPAR-1 has no known function in embryos (Monen et al., 2015). Because these orthologues share extensive DNA sequence identity, it is not possible to independently target them by RNAi; therefore, we will refer to their depletion as CENP-A RNAi (Monen et al., 2005, 2015). Topo-II also has two C. elegans orthologues (CIN-4 and TOP-2, both hits in our screen). However, the TOP-2 gene harbors the most extensive homology and, thus, was used in our studies. Long-chromosome embryos treated with partial CENP-A or topo-II RNAi (mimicking the levels from the screen) arrested with varying numbers of cells and appeared to exhibit chromosome loss independent of cell lineage or fate (Fig. S1, a–c). Thus, reducing the levels of CENP-A (and thus centromere chromatin) or topo-II resulted in lethality in C. elegans long-chromosome embryos compared with wild type.

Endogenous CENP-A and topo-II localize to distinct linear areas on mitotic holocentric chromosomes

Consistent with a structural role in chromosome condensation, CENP-A localizes linearly along mitotic chromosomes in C. elegans. However, topo-II localization has not previously been evaluated (Buchwitz et al., 1999). In monocentric organisms (e.g., mammals), topo-II localizes to chromosome arms and is concentrated at the condensed centromere regions, leading to the hypothesis that it serves as a structural element (Earnshaw et al., 1985; Taagepera et al., 1993; Rattner et al., 1996; Warsi et al., 2008). To test whether topo-II localized in a manner consistent with it serving as a structural element on C. elegans holocentric chromosomes, we generated an affinity-purified polyclonal antibody directed against C. elegans topo-II. Immunofluorescence staining with this antibody revealed linear localization along the exterior of mitotic chromosomes after nuclear envelope breakdown (NEBD; prometaphase; Fig. 2 a). The linear, exterior localization pattern is unlikely caused by a failure of the antibody to penetrate the chromatin, as other similarly generated antibodies can penetrate chromosomes prepared with identical methods (Oegema et al., 2001). Localization was lost after complete RNAi of topo-II, confirming that our RNAi is effective and the antibody specific (Fig. S1, d and e). Topo-II localization was confirmed using an endogenous superfolded GFP (sfGFP)–tagged version (the only copy in the animal, generated by genome editing; Fig. S2, a–c; Dickinson and Goldstein, 2016).

Figure 2.

Endogenous topo-II forms axes distinct from the centromere and the condensin complex. (a) One-cell embryos fixed and stained with anti–topo-II antibody and DAPI, at different mitotic stages. The insets show zoomed-in images of a single prometaphase (Prometa.) chromosome. Anti–topo-II antibody probing the whole-worm lysate by Western blotting (WB) is shown. The asterisk indicates a band that is unaffected by top-2 RNAi. (b) Centromeres marked with GFP–KNL-2 (OD31) or GFP-condensin (GFP–CAPG-2 and OD112) expressing single-cell embryos immunostained with anti–topo-II antibody to compare centromere and condensin with topo-II localization. The montage shows longitudinal views of a single prometaphase chromosome (first and third rows) and cross-sectional views of a chromosome (second and last rows). Insets show a zoomed-in cross-sectional view of a single chromosome. Schematics of a single prometaphase chromosome show a 3D view of spatial organization of topo-II (red), centromere (green), condensin (light blue), and DNA (dark blue). Bars: (fluorescence images) 1 µm; (brightfield images) 5 µm.

To further evaluate the roles of topo-II and CENP-A, we sought to determine whether topo-II localizes to chromosomes in a manner distinct from the centromere, which also forms a linear, exterior axis (Buchwitz et al., 1999). We fixed and stained embryos for CENP-A and topo-II and found that topo-II was excluded from centromeres (Fig. 2 b). These data support the hypothesis that topo-II and CENP-A are independent. The condensin complex localizes to and is required for assembly of mitotic chromosomes. In C. elegans, condensin localizes to a linear element on mitotic chromosomes. We costained topo-II and condensin (the SMC-4 subunit) and found distinct localization on mitotic chromosomes (Fig. 2 b). Thus, based on high-resolution analysis, topo-II localizes distinctly from CENP-A and the condensin complex on holocentric mitotic chromosomes.

CENP-A and topo-II contribute to chromosome length in opposing manners

Our screen suggested that CENP-A and topo-II contribute to setting proper chromosome length in some fashion. We predicted that depleting either protein would affect chromosome size in wild-type animals. Depletion (48 h RNAi) of either protein in C. elegans with wild-type chromosome length resulted in embryonic lethality stemming from zygotic condensation and segregation defects that preclude analysis of chromosome-length scaling during embryogenesis (Maddox et al., 2006). To characterize the cytological effects underlying the lethality observed in our screen, we partially depleted these proteins and analyzed single-cell embryos (Fig. 3 a and Table 2). We measured chromosome length and found that partial depletion of CENP-A resulted in shorter chromosomes compared with controls (Fig. 3 b and Table 1). In contrast, depletion of topo-II resulted in longer chromosomes (Figs. 3 b and S2 d and Table 1).

Figure 3.

CENP-A and topo-II work antagonistically to set chromosome size. (a) Representative images of single-cell embryos expressing H2B::mCherry and GFP::CENP-A (strain OD421; Fig. S2; Gassmann et al., 2012) and measurements of GFP::CENP-A intensity frequency distribution in control (black circle; n = 16), CENP-A RNAi (dark green triangles; n = 17), partial CENP-A RNAi (aqua squares; n = 23), topo-II RNAi (purple diamonds; n = 8), topo-II + partial CENP-A RNAi (pink stars; n = 3), rcc-1 (open purple circle; n = 8), or rcc1 + partial CENP-A RNAi (open blue squares; n = 7) conditions. Asterisks denote statistically different pairwise comparisons (see Table 2 for details). (b) Representative images of TH32 (animals of normal karyotype [six haploid chromosomes]) single-cell embryos containing GFP::histone-H2B (to visualize chromosomes) and GFP::γ-tubulin (centrosomes; to monitor mitotic progression) and frequency distribution of chromosome length measurements after control (black circles; n = 89), partial CENP-A RNAi (aqua squares; n = 84), topo-II RNAi (purple diamonds; n = 75), or topo-II + partial CENP-A RNAi (pink stars; n = 71). Ana., anaphase; Meta, metaphase; Prometa., prometaphase. Conditions are significantly different by one-way ANOVA. Error bars represent 95% confidence interval. Bars, 5 µm.

To dissect this functional interplay, we partially depleted CENP-A and topo-II simultaneously (Fig. 3 b). Double depletion rescued chromosome length in the early embryos nearly to control conditions (statistically different from each individual RNAi, Fig. 3 b and Table 1; and depletion quantification, Table 2). These animals displayed severe anaphase bridging defects (Fig. 3 b), indicating that the proteins were in fact depleted to a significant level. These findings suggest that CENP-A–containing chromatin elongates, whereas topo-II compacts holocentric chromosomes. Given that our screen was designed to identify proteins that promote chromosome shortening (as we have found topo-II to do), it was unexpected to find that CENP-A promotes chromosome elongation. We conclude that our screen was not as specific as intended and that hits can be thought of more broadly as involved in mitotic chromosome structure. It is not unheard of for two proteins with opposing functions to reveal similar cellular phenotypes (for instance, Rho GTPase-activating protein [GAP] or guanine exchange factor [GEF] depletion leads to failure of cytokinesis; unpublished data).

Table 1.Mean chromosome length measurements at the one-cell stage
Table 2.Mean chromatin CENP-A fluorescence intensity at the one-cell stage relative to control

Titration of CENP-A, but not topo-II, via nuclear trafficking is required for chromosome-size scaling during embryonic development

Previously, we showed that nuclear trafficking via the RAN pathway is required for regulating chromosome-size scaling in the C. elegans early embryo. We proposed a model wherein a chromosome compaction inhibitor is titrated inside the nucleus over the course of development (concomitant with reduced nuclear size), resulting in reduced chromosome length in smaller cells (Ladouceur et al., 2015). To test this hypothesis, we measured chromatin-associated CENP-A protein levels just after NEBD during various stages of early embryonic development. CENP-A chromatin levels decreased during development from the two- to eight-cell stage (Fig. 4 a; two- to eight-cell stage, P < 0.0001 [one-way ANOVA]; 16-cell stage and older, P = 0.3113 [one-way ANOVA]). This decrease essentially matched the rate of chromosome-size scaling that we reported previously (Ladouceur et al., 2015). In contrast, topo-II levels on chromatin did not vary during embryonic development until after the 50-cell stage, and depletion of topo-II did not affect CENP-A protein levels (Fig. S2 e; 2- to 49-cell stage, P = 0.5720 [one-way ANOVA]; 50-cell stage and older, P < 0.0001 [one-way ANOVA]; and Fig. 2 a and Table 2).

Figure 4.

Chromatin-associated CENP-A levels decrease during development and are regulated by nuclear import. (a) Representative images of embryos expressing GFP::CENP-A at three different developmental stages and frequency distribution of relative intensity measurements of chromatin GFP::CENP-A during development, from the 2- to 100-cell stage. Relative intensity measurements of chromatin GFP::CENP-A during development are different by one-way ANOVA. n =14 (2 cells), 18 (4 cells), 28 (8 cells), 12 (16 cells), 18 (30–49 cells), 24 (50–69 cells), and 9 (70–100 cells). Error bars represent mean and 95% confidence interval. (b) Representative images of two cell–stage embryos expressing GFP::CENP-A (OD421) after RNAi depletion of control (black circles; n = 72) or rcc-1 RNAi (purple circles; n = 68). Mean GFP::CENP-A relative intensities according to chromosome length are graphed (mean chromosome length measurements at 2-, 4-, 8-, and 16-cell stages taken from Ladouceur et al., 2015. Control conditions are different by one-way ANOVA (P < 0.001), and rcc1 RNAi conditions are not different by one-way ANOVA (P = 0.14). Bars, 5 µm.

Based on the above results, we hypothesized that partial depletion of the RanGEF RCC1 would disrupt CENP-A import (Ladouceur et al., 2015). Depletion of RCC1 led to reduced levels of CENP-A on chromatin that remained constant over the course of early development, compared with the control (Fig. 4 b; control, P < 0.0001 [one-way ANOVA]; RCC1 RNAi, P = NS [one-way ANOVA]). Double RCC1/CENP-A partial RNAi led to chromosome length not significantly different from each single RNAi (Fig. S2 f). In all cases, CENP-A levels scaled with chromosome size. However, we cannot exclude that other RAN pathway targets are critical to setting mitotic chromosome size. To test whether CENP-A import or CENP-A chromatin assembly is necessary for chromosome-length scaling, we blocked CENP-A nucleosome assembly, but not nuclear import, by depleting the centromere licensing factor KNL-2 (Maddox et al., 2007). Partial KNL-2 depletion (full depletion is lethal) reduced CENP-A chromatin without affecting CENP-A protein levels, concomitantly increasing unincorporated nuclear CENP-A (Fig. S1 c). Chromosomes in embryos partially depleted of KNL-2 were abnormally short, fitting with our data that CENP-A chromatin leads to longer chromosomes (Fig. S1 c). Together, these results support the idea that CENP-A chromatin is required for chromosome-length scaling during early C. elegans development.

CENP-A acts as a ruler for chromosome length

In C. elegans, CENP-A is incorporated and maintained in discrete domains distributed periodically along the genomic positional length of each chromosome (Oegema et al., 2001; Gassmann et al., 2012; Steiner and Henikoff, 2014). Because of this discontinuous distribution, CENP-A chromatin appears, via high-resolution light microscopy, as a patchy plate on the surface of metaphase holocentric chromosomes. CENP-A chromatin assembles during prophase to form a linear array on each chromosome, perhaps contributing to the rigidity of mitotic chromosomes (Maddox et al., 2006). In support of this idea, severe loss of CENP-A chromatin causes chromosomes to appear collapsed and round, instead of rod shaped (Oegema et al., 2001; Maddox et al., 2006). As a partial decrease of CENP-A chromatin resulted in abnormally short but still rod-shaped chromosomes, we hypothesized that mitotic self-association of dispersed CENP-A–containing chromosome loci contributes to chromosome morphology, with the quantity of centromeric DNA acting as a molecular ruler that sets chromosome length. To test this hypothesis, we plotted the amount of CENP-A on each fully condensed mitotic chromosome just before metaphase relative to chromosome length (Fig. 5, a and b). We found a linear relation between CENP-A levels and chromosome length, with shorter chromosomes containing less CENP-A than longer chromosomes (Fig. 5 b; control: y = 7.405 x ± 0.5 + 3.49 ± 3.49). This effect is mediated by centromeric chromatin, as depletion of the kinetochore protein CENP-C (whose depletion does not affect CENP-A levels in C. elegans) did not alter chromosome length (Maddox et al., 2007). By extension, this analysis reveals that the amount of CENP-A per unit length of condensed chromosome (not genetic distance) is constant in C. elegans.

Figure 5.

The amount of CENP-A incorporated into existing centromere domains sets the chromosome length. (a) Representative images of single-cell embryos expressing GFP::CENP-A and H2B::mCherry after control or CSR-1 RNAi. (b) GFP::CENP-A sum intensity for each individual segmented chromosome in relation to chromosome length and chromosome length for individual segmented chromosomes after control and csr-1 RNAi. Each slope is statistically different from zero. The slopes of the linear regression of the two conditions are statistically different. P < 0.01. (c) GFP::CENP-A maximum voxel intensity of individual chromosomes after control and CSR-1 RNAi. (b and c) All intensity values are relative to the maximum intensity of controls. Control (black circles), n = 104; and csr-1 RNAi (purple stars), n = 94 chromosomes measured in >10 embryos. (d) Representative SIM images of chromatin squashes of a single-cell GFP::CENP-A embryo after control RNAi. The arrow points to a representative focus measured in e. (e) Distribution plot of full-width at half-maximum intensity of individual GFP::CENP-A foci measured on images as in d for control (black; n = 21) and csr-1 RNAi (purple; n = 18). Box plots were generated with the application found on http://boxplot.tyerslab.com/. Centerlines show the medians. Box limits indicate the 25th and 75th percentiles as determined by R software. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. n = 21 and 18 sample points. (f) Schematic for the model of how CENP-A vs. topo-II impacts chromosome length and the model of centromere expansion vs. neo-centromere formation. Bars, 5 µm. *, P < 0.01.

If CENP-A epigenetic domains indeed set mitotic chromosome size, changing the amount of CENP-A chromatin should modify the ruler. In C. elegans embryos, CENP-A incorporation is inversely correlated to germline transcription (high transcription, low CENP-A incorporation; low transcription, high CENP-A; Gassmann et al., 2012; Steiner and Henikoff, 2014). We disrupted germline transcription by depleting the Argonaut protein CSR-1 (Claycomb et al., 2009) and thus increased the total amount of CENP-A incorporated in individual chromosomes in early embryos (Fig. 5 b). CENP-A overincorporation led to mitotic errors in the first division, precluding analysis in the resulting blastomeres. Interestingly, the linear relation between CENP-A chromatin levels and chromosome length was altered in CENP-A overincorporation in single-cell embryos (Fig. 5 b; control: y = 7.405 x ± 0.5 + 3.49 ± 3.49; csr-1 RNAi: y = 15.82 x ± 1.68 − 1.80 ± 12.92; differences in slope, P < 0.0001). These results are consistent with the hypothesis of an upper limit, or plateau, for chromosome length as reported for other mitotic structures (Schubert and Oud, 1997; Wühr et al., 2008; Ladouceur et al., 2015). In cells depleted of CSR-1, CENP-A was overincorporated compared with controls at the same developmental stage. As expected from a CENP-A ruler model, cells with increased CENP-A had longer chromosomes than controls (Fig. 5 b).

Centromere domain size, rather than domain number, defines C. elegans mitotic chromosome length

Chromatin immunoprecipitation sequencing analysis in C. elegans found that CENP-A localizes in broad chromatin domains with each chromosome possessing ∼100 domains spaced at distances ranging from 290 bp to 1.9 Mb (median of 83 kb), a distance that roughly fits the predicted loop size formed in mitotic chromosome assembly (Naumova et al., 2013; Steiner and Henikoff, 2014). In light of our measurements of decreased CENP-A levels throughout development, we developed two hypothesis: (1) either the number of CENP-A nucleosomes in each centromeric domain is larger in longer chromosomes (centromere expansion hypothesis) or (2) number of domains decreases over developmental time resulting in proportionally shorter chromosomes in smaller cells (neo-centromere hypothesis; Fig. 5 f). In the first case, individual, subresolution CENP-A domains would have an increased intensity in longer chromosomes, whereas in the second, individual domains would retain their intensity, but there would be more domains per chromosome in longer chromosomes. We measured CENP-A intensity of individual resolvable domains in conditions that resulted in altered CENP-A amounts (CSR-1 RNAi). We found an increase in CENP-A maximum fluorescent intensity per domain on individually segmented chromosomes in CSR-1 RNAi compared with control embryos (Fig. 5 c; control: 0.48 ± 0.01; CENP-A overincorporation: 0.91 ± 0.03). These results support the centromere expansion hypothesis for CENP-A loading.

CENP-A domains observed in the microscope do not represent bead-on-a-string level of chromatin and are more likely to be regions of GFP::CENP-A chromatin of a few kilobases or longer. To more precisely analyze individual domains, we performed prometaphase chromosomes squashes by squashing individually observed embryos (Fig. 5, d and e). We were unable to detect changes in CENP-A foci size or distribution per unit length of chromatin by confocal microscopy (not depicted) or structured illumination microscopy (SIM; Fig. S3 a; Gustafsson et al., 1999

Anonymous

Archived from groups: alt.games.nintendo.pokemon (More info?)

White Cat wrote:
> John Salerno wrote:
>> I'm wondering which of the two everyone prefers. Seems like the
>> benefit to Synthesis is that you always heal at least 1/2 HP
>> (assuming weather is normal), but with Giga Drain, you can do some
>> good damage and also heal, although maybe not quite as much. I
>> taught my Bulbasaur Giga Drain, so I don't want to replace it (and
>> lose it) if it's better than Synthesis.
>
> Are you talking about a link battle or playing the RPG?
>
> I'd definitely take Synthesis in a linkup, but it's not really
> necessary in the RPG, since you can use Potions. In a link battle,
> you might want both, since Giga Drain is one of the best offensive
> Grass moves now that Razor Leaf isn't an almost-always-critical-hit.
>
> -- White Cat
> http://pokedex.kary.ca/

Mainly just the single player game. I figured that might be the case, since
you can get through the game with all offensive moves. Besides, I just beat
the Elite Four last night, so it's not like it was a problem for me. Only
thing I don't like is that my Venusaur has all grass moves. Razor Leaf,
Frenzy Plant, Giga Drain, and Sleep Powder (grass, right?). I'm going to
replace Sleep Powder with Sludge Bomb when I get it, but any advice for the
other moves? I hate to get rid of Razor Leaf, it seems very useful. The
other two are irreplaceable, so I have to be careful there. I'm thinking
about using a PP UP on Frenzy Plant.