Studies on the high molecular weight form of polypeptide chain elongation factor-1 from pig liver. III. Temperature-dependent dissociation into subunits in the presence of GTP

Dissociation of highly purified EF-1 alpha beta gamma (a high molecular weight form of polypeptide chain elongation factor-1) from pig liver into EF-1 alpha and EF-1 beta gamma at various temperatures was examined and the following results were obtained. (i) When dissociation of EF-1 alpha beta gamma was analyzed by gel filtration with Sephacryl S-200, it was found that in the absence of GTP, it did not dissociate at any temperature between 4 and 37 degrees C, whereas in the presence of GTP, it tended to dissociate with elevation of the temperature, and almost complete dissociation was observed at 32 degrees C. This indicated that the dissociation constant of EF-1 alpha beta gamma into EF-1 alpha and EF-1 beta gamma in the presence of GTP increased with increase in the temperature. (ii) When gel filtration was performed in the presence of both GTP and [14C]Phe-tRNA, the formation of a ternary complex of EF-1 alpha . GTP . [14C]Phe-tRNA from EF-1 alpha beta gamma was noted, and its amount was found to increase with elevation of the temperature. (iii) The amount of [14C]Phe-tRNA bound to ribosomes dependent on added EF-1 alpha beta gamma similarly increased with increase in the temperature, as in the case of ternary complex formation, whereas the binding of [14C]Phe-tRNA to ribosomes dependent on free EF-1 alpha proceeded fairly well even at 0 degrees C. From these results we concluded that among the reaction steps in the binding of [14C]Phe-tRNA to ribosomes dependent on EF-1 alpha beta gamma, dissociation of EF-1 alpha beta gamma to form EF-1 alpha . GTP and EF-1 beta gamma in the presence of GTP is the step which is strongly influenced by temperature.     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Aminoacyl-tRNA-elongation factor Tu-ribosome interaction leading to hydrolysis of guanosine 5'-triphosphate

We investigated the elongation factor Tu (EF-Tu) dependent binding of Phe-tRNA and Phe-tRNAs with the nicks at positions 46, 37, and 17 to the Escherichia coli 70S ribosome-poly(U)-tRNAPhe complex. Binding of Phe-tRNA1-45 + 47-76, Phe-tRNA1-36 + 38-76, or Phe-tRNA1-16 + 17-76 to the 70S ribosome has been found to be poly(U) X tRNA dependent and, similar to that of intact Phe-tRNA, is inhibited by the antibiotic thiostrepton. We have further found that, contrary to a previous report [Modolell, J., Cabrer, B., Parmeggiani, A., & Vazquez, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1796], the EF-Tu-ribosome GTPase mediated by Phe-tRNA is not inhibited by thiostrepton; rather, the drug stimulates the endogenous GTPase of the EF-Tu X 70S ribosome. Phe-tRNA fragments 47-76, 38-76, and 17-76 all promote the EF-Tu X GTPase reaction in the presence of 70S ribosome-poly(U)-tRNAPhe yeast. Moreover, since the GTPase-promoting activities of both the short and long fragments are similar, it appears that the most important aminoacyl transfer ribonucleic acid (aa-tRNA) interaction with EF-Tu occurs alongside its 3' quarter. Thiostrepton slightly stimulates the GTPase activity of these Phe-tRNA fragments. Although the Phe-tRNA1-36 + 38-76 cannot bind to poly(U) during its binding to 70S ribosomes, its binding at high Mg2+ concentration occurs at the A site. Thus, most of the bound modified Phe-tRNA functions as the acceptor in the peptidyltransferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity.

The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.     

The use of glutaraldehyde fixation to analyze the mechanism of factor catalyzed reassociation of eukaryotic ribosomal subunits

Glutaraldehyde fixation was used to analyze the mechanism of reassociation of ribosomal subunits catalyzed by a factor in rat liver cytosol. Unstable 40S–60S couples formed spontaneously in buffer alone; the couples were dissociated by hydrostatic pressure during centrifugation unless they were fixed with glutaraldehyde. Increased numbers of stable 80S ribosomes were formed in the presence of poly (U), Phe-tRNA and G-25 fraction (which contains the initiation factor EIF-1). The factor would seem then to both increase formation of 80S ribosomes and stabilize the monomer. An additional effect of the factor is to inhibit the formation of the unstable 40S–60S couples which form in the presence of Phe-tRNA alone.     

Egg-laying preference of female fungus gnat Bradysia sp. nr. coprophila (Diptera: Sciaridae) on three different soilless substrates

Fungus gnats, Bradysia spp., are major insect pests in greenhouses. Adult female fungus gnats prefer to lay eggs in growing medium that is microbially active or that contains high amounts of peat moss or hardwood bark. However, egg-laying preference has not been demonstrated quantitatively. This study was designed to determine whether fungus gnat Bradysia sp. nr. coprophila females prefer any of the three soilless growing media provided. The three soilless growing media tested were Metro-Mix 560 with Scott's Coir, Sunshine LC1 Mix, and Universal SB 300 Mix. Initially, the egg-laying potential of the fungus gnat species used in this study was assessed by dissecting mated females after 24, 48, and 72 h. For the egg-laying preference experiment, adults that emerged from pupae were aspirated into a plastic vial, sexed, and then allowed to mate for 24 h. Individual mated females were released into an experimental chamber (15 by 15 by 5-cm plastic container) consisting of four 6-cm petri dishes, three of which contained soilless growing media and one with filter paper (control). In total, there were 50 experimental chambers, with each chamber representing a replication. Females remained in the experimental chambers for 48 h after which the growing media were processed using a flotation/extraction method. The number of eggs laid by female fungus gnats ranged from 21 to 217 with most eggs recovered after 48 h (141.0 +/- 9.3). There were no significant differences among the three soilless growing media in terms of number of eggs laid, although all three growing media were significantly different from the filter paper with higher numbers of eggs laid in the soilless growing media than the filter paper. Despite no significant difference among the growing media in the number of eggs laid, fungus gnat females tended to lay eggs more often, based on the number of petri dishes in which at least one egg was laid, in Metro-Mix 560 (86%) than Sunshine LC1 (66%), Universal SB 300 (52%), or filter paper (18%). Based on the results of this study, female fungus gnats may not prefer a specific growing medium for oviposition. However, fungus gnat females may rely on other factors not tested in this study such as moisture content and volatiles emitted from growing media in their decision where to lay eggs.     

Effects of nucleotide- and aurodox-induced changes in elongation factor Tu conformation upon its interactions with aminoacyl transfer RNA. A fluorescence study

The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base. The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex. The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions. In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome. Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites in the tRNA.

Trans-diamminedichloroplatinum (II) was used to induce reversible crosslinks between EF-Tu and Phe-tRNA(Phe) within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex. Up to 40% of the complex was specifically converted into crosslinked species. Two crosslinking sites have been unambiguously identified. The major one encompassing nucleotides 58 to 65 is located in the 3'-part of the T-stem, and the minor one encompassing nucleotides 31 to 42 includes the anticodon loop and part of the 3'-strand of the anticodon stem.     

Properties of phenylalanine transfer ribonucleic acid with modified 3''-terminal end in protein biosynthesis using a rabbit reticulocyte cell-free system: effect of the replacement of cytidine residues from the CpCpA end of tRNA by 5-iodocytidine or 2-thiocytidine.

Phe-tRNA Phe from yeast containing 2-thiocytidine or 5-iodocytidine in position 75 of the polynucleotide chain or Phe-tRNA Phe in which both positions 74 and 75 were substituted by 5-iodocytidine were investigated in the poly U-dependent polyphenylalanine synthesis on ribosomes from rabbit reticulocytes. Phe-tRNA Phe-Cps2CpA was nearly as active as the native Phe-tRNA Phe-CpCpA in the overall process. Phe-tRNA Phe-Cpi 5CpA as well as Phe-tRNA Phe-i5Cpi 5CpA were considerably less active than the native species. Investigation of individual steps of protein biosynthesis with these modified substrates revealed that the donor activity of peptidyl-tRNAs which contain 5-iodocytidine in their 3'-terminus is strongly imparied suggesting exacting structural requirements for the interaction of the CpCpA end of tRNA with the ribosomal P-site.     

Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes

Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-1H, is a high-molecular-weight aggregate (M_r > 500,000) containing four distinct polypeptides (α, β, γ, δ). The other form, EF-1α, consists of a single polypeptide which is the same as the α subunit of EF-1H. Both EF-1α and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)-directed polyphenylalanine synthesis. The activity of EF-1α is enhanced in polyphenylalanine synthesis by a complementary component, EF-1βδ. It is also shown that EF-1βδ can facilitate an exchange of EF-1α-bound GDP for GTP. The EF-1α dissociation constants for GDP and GTP were 0.47 and 0.55 μm respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 μm, respectively. Thus, while EF-1α and EF-1H had approximately the same affinities for GDP and GTP, the EF-1α dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-1α or EF-1H with GTP and Phe-tRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along with studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes.     

The effect of removal or replacement with proflavine of the Y base in the anticodon loop of yeast tRNAPhe on binding into the acceptor or donor sites of reticulocyte ribosomes

Phe-tRNA from yeast has a highly modified nucleoside, called Y, adjacent to the 3′ side of its anticodon, that can be removed or replaced with proflavine. In a protein-synthesizing system from rabbit reticulocytes, poly (U)-directed binding and polyphenylalanine synthesis are low with these modified Phe-tRNA species relative to the corresponding values with unmodified Phe-tRNA. However, polymerization can be increased with relatively large amounts of elongation factor I. The modified Phe-tRNA species bound to the ribosomes with poly(U) either in the presence or absence of elongation factor I and GTP is immediately reactive in the peptidyl transferase reaction measured by the formation of diphenylalanine or phenylalanyl-puromycin. It appears to have been bound directly into the donor ribosomal site by either the nonenzymatic mechanism involving Mg²⁺ or by the enzymatic mechanism involving EF-I and GTP.     

Variation of taxane content in needles of Taxus x media cultivars with different growth characteristics

Needles from 17 different Taxus x media cultivars, belonging to 4 groups showing different growth characteristics, were analyzed using high performance liquid chromatography for their content of 10-deacetylbaccatin III, baccatin III, cephalomannine and paclitaxel (Taxol). The 4 Taxus x media cultivar groups were: 1.) medium to fast growing and upright form; 2.) slow growing and upright form; 3.) fast growing and spreading form; and 4.) slow growing and spreading form. The purpose of this study was to identify yew cultivars of fast growth rate, upright growth and high taxane content in their needles. The highest content of paclitaxel was found in 'Coleana' of group 1 (378 microg/g of the extracted dry weight). Three cultivars in group 1, 'Coleana', 'Stovekenii' and 'Hicksii', make good candidates for taxane extraction because of their high paclitaxel and 10-deacetylbaccatin III content, fast biomass accumulation and upright growing form. They are also good starting materials to develop alternative methods for the production of paclitaxel and its analogous compounds through modern biotechnology approaches.     

Translational accuracy enhanced in vitro by (p)ppGpp

Summary The effect of (p)ppGpp on the accuracy of translation in vitro was studied with a system that has a missense error frequency similar to that of living bacteria. When poly (U)¹ is translated, limiation of the system in Phe increases the Leu missense error frequency. The introduction of (p)ppGpp to the Phelimited mixtures reduces significantly the missense errors as well as reduces the rate of translation. The introduction of (p)ppGpp to a full system has no effect on the accuracy of translation but does reduce its rate. The effects of (p)ppGpp on rate and accuracy of translation can be simulated in part by other inhibitors of translation such as GDPCP, fusidic acid and tetracycline. Furthermore, the presence of ppGpp or GDPCP in a Phe-limited system leads to an accumulation of Phe-tRNA, while a Phe-limited system that contains only GTP has negligibly small concentrations of Phe-tRNA. We conclude that one way in which (p)ppGpp improves the accuracy of translation is by permitting the system to maintain a favorable Phe-tRNA/Leu-tRNA ratio.     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

Phenylalanyl-tRNA synthetase editing defects result in efficient mistranslation of phenylalanine codons as tyrosine

Translational quality control is monitored at several steps, including substrate selection by aminoacyl-tRNA synthetases (aaRSs), and discrimination of aminoacyl-tRNAs by elongation factor Tu (EF-Tu) and the ribosome. Phenylalanyl-tRNA synthetase (PheRS) misactivates Tyr but is able to correct the mistake using a proofreading activity named editing. Previously we found that overproduction of editing-defective PheRS resulted in Tyr incorporation at Phe-encoded positions in vivo, although the misreading efficiency could not be estimated. This raised the question as to whether or not EF-Tu and the ribosome provide further proofreading mechanisms to prevent mistranslation of Phe codons by Tyr. Here we show that, after evading editing by PheRS, Tyr-tRNA(Phe) is recognized by EF-Tu as efficiently as the cognate Phe-tRNA(Phe). Kinetic decoding studies using full-length Tyr-tRNA(Phe) and Phe-tRNA(Phe), as well as a poly(U)-directed polyTyr/polyPhe synthesis assay, indicate that the ribosome lacks discrimination between Tyr-tRNA(Phe) and Phe-tRNA(Phe). Taken together, these data suggest that PheRS editing is the major proofreading step that prevents infiltration of Tyr into Phe codons during translation.     

Inhibition of deacylation and improvement in N-hydroxysuccinimide ester modification of phenylalanyl-tRNA by dimethyl sulfoxide.

We have found that dimethyl sulfoxide significantly inhibits deacylation of Phe-tRNA. This allows a high pH in N-hydroxysuccinimide ester reactions while maintaining a high level of aminoacylated tRNA, improving the overall yield of the Phe-tRNA modification reaction.     

Elongation factor Tu-induced conformational changes of ribosomes detected by iodination

The effect of elongation factor (EF) Tu, bound to the ribosome with the help of poly(uridylic) acid, Phe-tRNA and guanyl-5-yl methylene diphosphonate, on the conformation and/or chemical environment of ribosomal proteins has been examined using, as a probe, protein iodination. Ribosomes complexed only with poly(uridylic acid) and Phe-tRNA have been used as a control. EF-Tu on the ribosome significantly increases the iodination of proteins S7, S10 and L3 and decreases that of S21 and L18.     

A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein

A single amino acid substitution (Asp to Asn) at position 138 of Escherichia coli elongation factor Tu (EF-Tu) was introduced in the tufA gene clone by oligonucleotide site-directed mutagenesis. The mutated tufA gene was then expressed in maxicells. The properties of [35S]methionine-labeled mutant and wild type EF-Tu were compared by in vitro assays. The Asn-138 mutation greatly reduced the protein's affinity for GDP; however, this mutation dramatically increased the protein's affinity for xanthosine 5'-diphosphate. The mutant protein forms a stable complex with Phe-tRNA and xanthosine 5'-triphosphate, which binds to ribosomes, whereas it does not form a complex with Phe-tRNA and GTP (10 microM). These results suggest that in EF-Tu.nucleoside diphosphate complexes, amino acid residue 138 must interact with the substituent on C-2 of the purine ring. Thus, in wild type EF-Tu, Asp-138 would hydrogen bond to the 2-amino group of GDP, and in the mutant EF-Tu, Asn-138 would form an equivalent hydrogen bond with the 2-carbonyl group of xanthosine 5'-diphosphate. Aspartic acid 138 is conserved in the homologous sequences of all GTP regulatory proteins. This mutation would allow one to specifically alter the nucleotide specificity of other GTP regulatory proteins.     

Aminoacyl-tRNA exclusively in the 3'-isomeric form is bound to polypeptide chain elongation factor Tu

2' and 3'-O-(N-acetyl-L-phenylalanyl)adenosine (Ac-Phe-Ado) were chemically synthesized. These two isomers were clearly separated from each other by high-performance liquid chromatography (HPLC). From the two isomers of [3H]Phe-tRNA in equilibrium, Ac-[3H]Phe-Ado was prepared, without any change in the 2'/3'-isomer ratio, by acetylation of the phenylalanyl residue with acetic anhydride followed by digestion with pancreatic RNase A. By HPLC analysis of this preparation of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was found to be 0.20:0.80. Further, [3H]Phe-tRNA was bound to Escherichia coli polypeptide chain elongation factor Tu (EF-Tu) with the ligand of GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GMP-P(NH)P). The ternary complex was treated with phenol and acetic anhydride, and then digested with pancreatic RNase A. By HPLC analysis of Ac-[3H]Phe-Ado, the abundance ratio of the 2'-isomer and the 3'-isomer of [3H]Phe-tRNA was determined to be 0.07:0.93 in the complex with EF-Tu.GTP and 0.04:0.96 in the complex with EF-Tu.GMP-P(NH)P. These results clearly indicate that the 3'-isomer, rather than the 2'-isomer, of aminoacyl-tRNA is exclusively involved in the ternary complex.     

Protein and nitrate content of lemna sp. As a function of developmental stage and incubation temperature

Lemna protein per frond and per root increases with developmental stage until plants are at least two generations old. Protein per frond, per root, and per unit dry weight is greater in plants grown at 23.9 C than at 18.3 C. More protein is found in fronds than in roots, and more nitrate occurs in roots than in fronds. Nitrate per root increases with developmental stage and is higher (per root) in plants grown at 23.9 C than in those grown at 18.3 C. The distribution of generations within a growing population is constant for at least eight doubling times. Whether populations multiply slowly at 15.6 C or more rapidly at 23.9 C, fronds which have not yet produced progeny form 62% of the population; fronds which are one generation old form 24% of the population; and fronds which are two generations old form 9% of the population.