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The mitochondrial gene encoding yeast cytochrome oxidase subunit II (Cox2p) specifies a precursor protein with a 15-amino-acid leader peptide. Deletion of the entire leader peptide coding region is known to block Cox2p accumulation posttranscriptionally. Here, we examined in vivo the role of the pre-Cox2p leader peptide and the mRNA sequence that encodes it in the expression of a mitochondrial reporter gene,ARG8m , fused to the 91st codon ofCOX2. We found within the coding sequence antagonistic elements that control translation: the positive element includes sequences in the first 14 codons specifying the leader peptide, while the negative element appears to be within codons 15 to 91. Partial deletions, point mutations, and local frameshifts within the leader peptide coding region were placed in both thecox2::ARG8m reporter and inCOX2 itself. Surprisingly, the mRNA sequence of the first six codons specifying the leader peptide plays an important role in positively controlling translation, while the amino acid sequence of the leader peptide itself is relatively unconstrained. Two mutations that partially block translation can be suppressed by nearby sequence substitutions that weaken a predicted stem structure and by overproduction of either the COX2 mRNA-specific translational activator Pet111p or the large-subunit mitochondrial ribosomal protein MrpL36p. We propose that regulatory elements embedded in the translated COX2 mRNA sequence could play a role, together with trans-acting factors, in coupling regulated synthesis of nascent pre-Cox2p to its insertion in the mitochondrial inner membrane.
Mitochondrial genetic systems have evolved to supply a limited number of subunits of enzymes that carry out respiration and oxidative phosphorylation in the inner membrane. These subunits, encoded in mitochondrial DNA (mtDNA), are assembled with imported products of nuclear genes by mechanisms that are poorly understood but genetically complex, at least in Saccharomyces cerevisiae (18, 19, 54). It seems likely that the expression of mitochondrial and nuclear genes that encode subunits of energy-transducing enzymes is coordinated with the assembly of those enzymes, although no clear mechanisms for this have been elucidated.
In this study, we have focused closely on expression of the mitochondrial gene encoding cytochrome c oxidase subunit II, Cox2p. Like most, if not all, yeast mitochondrial genes,COX2 is expressed under the control of a nuclearly encoded inner-membrane-bound mRNA-specific translational activator protein, Pet111p (14, 17). Pet111p is tightly associated with the inner mitochondrial membrane and is present at levels that limitCOX2 expression (17). This translational activator functionally interacts with the COX2 mRNA 5′ untranslated leader (5′-UTL) and promotes translation by an unknown mechanism (31, 39) that is conserved in other budding yeasts (10). Translation of other coding sequences artificially fused to the COX2 mRNA 5′-UTL is also activated by Pet111p (6, 17, 32), indicating that open reading frame sequences do not play a role in this activation step. Furthermore, its interaction with the COX2 mRNA 5′-UTL appears to be important for correct localization of Cox2p synthesis within the organelle (43).
Cox2p is synthesized as a precursor with a 15-amino-acid leader peptide (41, 46) that is cleaved in the intermembrane space after translocation of the amino terminal domain of pre-Cox2p through the membrane (4, 37, 41, 44). Translocation of the amino-terminal domain is not well understood mechanistically but depends on the highly conserved inner membrane protein Oxalp (5,21, 22, 27) and is thought to be cotranslational (14,40). While the leader peptide causes membrane association of a soluble passenger protein fused to it, it does not function as a classical signal sequence since it is incapable of directing translocation of the passenger protein through the inner membrane (21). Nucleic acid sequence comparisons indicate that the pre-Cox2p leader peptides of various budding yeast species, includingNeurospora crassa, and plants are not highly conserved (20, 25). Mammalian Cox2p entirely lacks a leader peptide (3, 50).
We previously began a study of the function of the pre-Cox2p leader peptide by deleting the codons that specify it from the S. cerevisiae COX2 mitochondrial gene (53). This deletion dramatically reduced the accumulation of Cox2p and caused a severe respiratory defect, but did not affect COX2 mRNA levels. The defect was bypassed by a chimeric gene whose product had the amino-terminal 251 residues of cytochrome b fused to the remaining COX2 coding sequence (53). However, the mechanism by which the deletion prevented COX2expression was not established: it could have affected translation, membrane insertion, or both.
Here, we examine in more detail the in vivo function of the pre-Cox2p leader peptide and the mRNA sequence that encodes it by site-directed mutation of the mtDNA sequence. We determine the effects of each mutation on functional expression of COX2, which demands synthesis and assembly of cytochrome oxidase, and on expression of a reporter gene, ARG8m (fused to the 91st codon of the COX2 reading frame), which depends only on translation of the chimeric mRNA. Surprisingly, we find that in the context of the COX2 coding sequence, the mRNA sequence encoding the leader peptide plays an important role in controlling translation, while the amino acid sequence of the leader peptide itself is relatively unconstrained. Our analysis suggests the existence of positive and negative regulatory elements embedded in the translated mRNA sequence specifying the N-terminal portion of pre-Cox2p, which could play a role in coupling regulated synthesis of the nascent Cox2p precursor to its insertion in the inner membrane.
Strains, media, and genetic techniques.All strains used in this study are listed in Table 1, with the exception of DFS160p0 (MATα leu2Δ arg8ΔURA3 ura3-52 kar1-1 ade2-101 [rho 0]) (6,49). Strain backgrounds were either D273-10B (ATCC 25627) or DBY947 (36). NB58 comes from the cytoduction ofcox2-20 mitochondria (53) into the [rho 0] derivative of NB40-3C.cox2-62 and cox2-60 are deletions of sequences −295 to +363 (11) and −63 to +66 (6) relative to AUG, respectively. Fermentable complete medium was YPDA or YPGalA: 1% yeast extract, 2% Bacto-Peptone, 100 mg of adenine/liter, and either 2% glucose or 2% galactose supplemented with 0.1% glucose. Nonfermentable medium was YPEG: 1% yeast extract, 2% bacto-peptone, 100 mg of adenine/liter, 3% ethanol, 3% glycerol. Minimal medium (0.67% yeast nitrogen base without amino acids) was supplemented with 2% glucose and specific amino acids, with uracil and adenine as required. Standard genetic methods were as previously described (15, 42). Yeast nuclear transformation was carried out using the one-step transformation method (8) for both library DNA and the plasmids YEp352 (23), pJM20 (33), pJM57 (31), or pNB107 (see below).
Mutagenesis of the leader peptide coding sequence.TheCOX2 plasmid pNB69 and thecox2::ARG8m plasmid pNB81, which also contains a marker fragment of COX3, were generated previously (6). They contain two unique sites surrounding the leader peptide coding sequence: PacI at −60 relative to the first COX2 codon and NsiI at +65. These sites were used to replace the wild-typePacI-NsiI fragment by mutant versions of theCOX2 leader peptide sequence. Novel mutagenized fragments were created by a two-step PCR (24) using low-error-rate DeepVent polymerase (NE Biolabs, Inc.). We first cloned thecox2-22 PacI-NsiI fragment into thePacI and NsiI sites of both pNB69 (to yield pNB82) and pNB81 (to yield pNB90). We subsequently used pNB82 and pNB90 as backbones in which to insert the other mutagenizedPacI-NsiI fragments since cox2-22introduces a unique SnaBI site between the PacI and NsiI sites (see Fig. 5A), allowing improved cloning efficiency by digestion of the ligation reaction products withSnaBI. Alternatively, some novel mutations were generated by the megaprimer method (45) using Taq polymerase (Boehringer, Inc.). In this case, the template was pNAB1, which contained the COX2 gene in pBKSII+ (Stratagene, Inc.) as aHindIII-BamHI fragment. All final plasmid constructs were confirmed by DNA sequencing using the primer COX2A, which was complementary to COX2 positions −254 to −237 relative to AUG.
Mitochondrial transformation and genetic manipulations.Plasmids carrying modified COX2 sequences were introduced into the [rho 0] mitochondria of strain DFS160p0 (Table 1) by high-velocity microprojectile bombardment (7, 56). Mitochondrial synthetic [rho −] transformants were identified, and their altered COX2 sequences were inserted into [rho +] mtDNA by recombination and selection of haploid cytoductants as previously described (6).cox2 mutants were generated by crossing synthetic [rho −] transformants with either [rho +] cox2-60 (NB97) orcox2-62 (NB40-3C) strains, andcox2::ARG8m mutants were generated by crossing synthetic [rho −] transformants with a [rho +]cox2-60::ARG8m strain (NB54) or by recombination with [rho +] cox2mutant strains. Haploid cytoductants were isolated unless otherwise indicated. In several cases, we also constructed the desired [rho +] strain by directly transforming [rho +] strains (6, 26) carrying the cox2-60 deletion (NB66 or NB104). This approach allows a quicker isolation of desired [rho +] strains, whose phenotypes were identical to those of corresponding mutants constructed by the two-step method. All mitochondrial mutations were verified by DNA sequencing (carried out by the Synthesis and Sequencing Facility in the Cornell Biotechnology Building) following integration into [rho +] mtDNA and PCR amplification from total DNA, as previously described (6).
Selection of suppressors and disruption of MRPL36. Spontaneous revertants of independent subclones of cox2-22strain NB64, cox2-27 strain NB140 and cox2-43strain NB178, were selected on YPEG medium after 1- to 4-week incubations at 28 or 16°C. Revertant phenotypes were confirmed by streak purification. The COX2 upstream region of revertant mtDNA was sequenced as described above. Revertants ofcox2-22 were genetically analyzed by two crosses. First, the spontaneous revertants were crossed to a nuclearly wild-type [rho 0] strain. Second, [rho 0] derivatives of the revertants were generated (15) and crossed with cox2-22 strain NB68. In both cases, the respiratory ability of the diploids was scored. Taken together, the results distinguish nuclear dominant, nuclear recessive, and mitochondrially inherited suppressor mutations.
Dosage-dependent nuclear suppressors of cox2-22 were isolated by transforming strain NB64 with an S. cerevisiaegenomic library in the multicopy plasmid YEp13 (35). Roughly 6,000 Leu+ transformants were scored for growth on YPEG medium at 28°C. Eleven such transformants exhibited cosegregation of Leu+ and Pet+ phenotypes after growth under nonselective conditions. Plasmids from these transformants were analyzed by partial sequencing of the inserts by using a YEp13-specific primer. Nine overlapping inserts were from chromosome II (pNB104-type), and two overlapping inserts were from chromosome XIII (pNB105-type). Only the pNB104-type plasmids which were found to also suppress cox2-27 were further analyzed to identify the suppressing gene.
The MRPL36 gene was isolated by cloning the 1.2-kbXhoI-SacI fragment from pNB104/40 into theSalI and SacI sites of YEp352 (23) to yield pNB107. MRPL36 was largely deleted by replacing the 0.65-kb internal BglII-KpnI fragment of pNB107 with the BamHI-KpnI URA3-containing fragment from pNB108 (pNB108 is the 1.56-kb URA3-containingNsiI fragment from YEp352 that was blunt ended and cloned in a YEp351 SmaI site). The resulting plasmid, called pNB109, carried a deletion of the whole MRPL36 coding sequence but the first eight codons. The wild-type strain NB80 was transformed with the 2.1-kb gel-purified PstI-SacI fragment of pNB109, and Ura+ transformants were selected. The disruption was confirmed by PCR analysis and at the genetic level by analysis of the linkage between the deletion marker URA3 andLYS2, a gene tightly linked to MRPL36. Strains carrying mrpL36Δ::URA3 were found to be [rho −] or [rho 0] by crossing to a nuclearly wild-type [rho 0] strain.
Analysis of cellular RNA and proteins.Total RNA was prepared and analyzed as previously described (11) except that the samples were supplemented with ethidium bromide before loading 10 μg in each lane. The COX2 probe was the 1.6-kbPacI fragment from pJM2 (32). Thecox2::ARG8m probe was the 1.4-kbPacI-BamHI fragment from pNB81, which contained part of the COX2 gene (approximately from the start of the mature mRNA to codon 91) and the wholeARG8m coding sequence. 15S rRNA was probed with either the 2-kb BamHI fragment from plasmid pYJL25 (52) provided by O. Groudinsky orXhoI-linearized plasmid pT82 (47).
Total cellular protein was extracted (58) from cells grown in YPGalA medium to a mid-exponential phase. Fifty to 200 μg of total protein, as indicated, were separated on 10 to 12% polyacrylamide gels and probed with monoclonal anti-Cox2p antibodies (obtained from Molecular Probes Inc.; provided by G. Dujardin) diluted to 1/5,000, CCO6 (38) (obtained from T. Mason) diluted to 1/50, or polyclonal anti-Arg8p antibody (49) diluted to 1/1,000. The monoclonal anti-outer membrane porin antibody from Molecular Probes was provided by G. Dujardin and was diluted to 1/5,000. Secondary anti-mouse or anti-rabbit antibodies were detected using the Pierce Inc. chemiluminescent substrate.
In vivo pulse labeling with [35S]methionine in the presence of cycloheximide was performed as previously described (15), with the following modifications. Cells were grown initially in liquid 1% yeast extract–2% Bacto Peptone–2% raffinose and then transferred to synthetic complete medium lacking Met (0.67% yeast nitrogen base, 0.08% CSM-Met [Bio 101, Inc.], 2% raffinose). Cells were labeled with 5 μCi of [35S]methionine (1,175 Ci/mmol; NEN, Inc.) for 10 min and then chased with unlabeled 2.5 mM methionine for 10 min before chilling and freezing. Crude mitochondria were isolated after the conversion of cells to spheroplasts as previously described (58), except that disruption was by vortexing with glass beads.
Deletion of pre-Cox2p leader peptide coding sequence dramatically decreases translation.We previously made 13- and 14-codon deletions of mtDNA specifying the pre-Cox2p leader peptide (allelescox2-20 and cox2-21, respectively) to generate genes that would encode a leaderless Cox2p protein similar to that of animals. We observed that both mutations dramatically decreased Cox2p accumulation and prevented respiratory growth, without altering the level of COX2 mRNA (53). This could have been due to decreased stability of the protein, decreased translation of the mRNA, or both. To distinguish the effects of the leader peptide deletions on Cox2p synthesis from those on Cox2p function, we took advantage of a synthetic mitochondrial reporter gene,ARG8m , which specifies a soluble enzyme necessary for arginine biosynthesis in the mitochondrion (49) by fusing it to the COX2 reading frame (6). Thiscox2::ARG8m chimeric gene comprises the first 91 codons of COX2, fused to codon 2 of ARG8m . When inserted precisely in place of the native COX2 gene in the mitochondrial genome,cox2::ARG8m complements the Arg− growth phenotype of a nuclear arg8mutation and directs the synthesis of a chimeric fusion protein. However, since the ARG8m sequence used here specifies the cleavage site for removal of the matrix targeting signal of pre-Arg8p, this chimeric Cox2p-Arg8p fusion protein is largely processed to yield mature Arg8p (6). Thus, translation of the chimeric cox2::ARG8m mRNA can be assayed by scoring for Arg+ growth independently of functional constraints on the structure of Cox2p.
Both of the 13- and 14-codon deletion mutations (cox2-20and cox2-21, respectively) were inserted into thiscox2::ARG8m reporter gene and placed into the mitochondrial genome by transformation and homologous recombination (see Materials and Methods). Neither thecox2-20::ARG8m norcox2-21::ARG8m construct complemented a nuclear arg8 mutation at the level of growth or directed synthesis of Cox2p-Arg8p, as measured by pulse labeling (Fig. 1; data not shown). Furthermore, neither accumulated detectable steady-state levels of reporter protein (Fig. 2). The lack of Arg8p synthesis was not due to an mRNA synthesis or stability defect, since the chimeric mRNA steady-state level was similar in the mutant and wild-type strains (Fig. 2).
It was unlikely that the leader peptide deletion mutants were lacking the Arg8p reporter protein due to degradation of the chimeric protein, since cleavage of the pre-Arg8p matrix targeting sequence should release mature, wild-type Arg8p from the fusion protein, as observed previously (6). Furthermore, we experimentally verified that a mutation causing instability of Cox2p did not prevent the accumulation of Arg8p encoded by a chimeric gene. A mutation that altered the pre-Cox2p leader peptide processing site from ND to KT (cox2-24) in an otherwise wild-type COX2 gene caused a severe respiratory defect and greatly reduced the level of Cox2p (Fig. 1 and 3C). However, when this mutation was inserted into thecox2::ARG8m reporter gene, the resulting strain was fully Arg+, synthesized pulse-labeled Cox2p-Arg8p (Fig. 1), and contained a normal steady-state level of the processed Arg8p reporter protein (not shown). Thus, the defect in pre-Cox2p processing destabilized Cox2p but had no effect on Arg8p translation, confirming that ourcox2::ARG8m reporter system can discriminate between gene expression defects caused by Cox2p instability and those caused by reduced translation.
Taken together, these findings demonstrate that deletion of the pre-Cox2p leader peptide coding sequence fromcox2::ARG8m (cox2-20::ARG8m andcox2-21::ARG8m ) produces mRNAs that are poorly if at all translated. This result is very surprising since in the complete absence of COX2 codons [the cox2(UTL)::ARG8m allele of strain HMD22], ARG8m is efficiently expressed from the COX2 locus (Fig. 1). Thus, the presence ofCOX2 codons 15 to 91 in the chimeric mRNA specified by the cox2-20::ARG8m allele apparently blocks translation of the downstreamARG8m sequence. On the other hand, fusion of theCOX2 codons 1 to 16 to a variant ofARG8m lacking its matrix targeting signal did not prevent the expression of the reporter (21). Taken together, these results suggest the existence of an element in the sequence of codons 15 to 91 that inhibits translation and a compensating positively acting element within the first 14 codons that specify the leader peptide.
Mutations within the leader peptide coding region that reduce downstream translation.To define more precisely the leader peptide coding sequences necessary for efficient translation of theCOX2 and cox2::ARG8m mRNAs, we constructed a set of mutants harboring small deletions and/or point mutations. In all cases, the initiation codon and the codons for residues 15 and 16 surrounding the leader peptide cleavage site (ND) were left unchanged. First, three small deletions spanning the remaining 13 codons were created: deletion of codons 2 to 6 (residues LDLLR, allele cox2-27), deletion of codons 7 to 10 (residues LQLT, allele cox2-23), and deletion of codons 11 to 14 (residues TFIM, allelecox2-28). None of these deletions had a significant effect on the steady-state levels of either the COX2 orcox2::ARG8m mRNAs (Fig. 3B and data not shown), and neither the codons 7-to-10 nor the codons 11-to-14 deletions detectably affected either respiratory growth or arginine prototrophy (Fig. 3A). In contrast, the codons 2-to-6 deletion, cox2-27, caused decreased respiratory growth at 28°C, prevented arginine prototrophy when inserted into the reporter construct (Fig. 3A), and reduced the steady-state level of Cox2p (Fig. 3C). Both of the growth phenotypes were enhanced when the incubation temperature was lowered to 16°C and were partially suppressed by increasing the temperature to 36°C (not shown). We also deleted codons 7 to 14 in a single mutation (cox2-35) (Fig. 3A). This eight-codon deletion mutation allowed nearly wild-type growth on both nonfermentable medium and minimal medium lacking arginine. These results demonstrate that codons 2 to 6 are most important for allowing the translation of downstream sequences. However, since deletion of these codons does not produce a phenotype as severe as the 13-codon deletion of cox2-20, codons 7 to 14 also contribute to the action of the putative positive element. In addition, these results demonstrate that the leader peptide can be shortened considerably without destroying pre-Cox2p function.
The cox2-22 mutation is a compound allele with both a deletion of codons 7 to 10 (cox2-23) and the translationally silent change of codon 6 from AGA to CGT (cox2-29), both of which specify R (48). Neither cox2-29 alone nor cox2-23 alone affected respiratory growth or arginine prototrophy (Fig. 3A). However, the combination of the two alterations in cox2-22 produced phenotypes similar to that of the codons 2-to-6 deletion,cox2-27, leaky respiratory and Arg+ growth at 28°C (Fig. 3A) and reduced Cox2p levels (Fig. 3C). Both phenotypes were enhanced by growth at a lower temperature and suppressed by growth at higher temperatures (not shown), as in the case ofcox2-27. The similarity of the cox2-22 andcox2-27 phenotypes suggests that both mutations could affectCOX2 translation by the same mechanism. Interestingly, since the only difference between the phenotypically silent codons 7-to-10 deletion (cox2-23) and cox2-22 was the translationally silent AGA-to-CGT alteration, these results suggest that the mRNA sequence could be important for the function of the putative positive element, independently of the leader peptide amino acid sequence it encodes.
Suppression of mutations within the leader peptide coding region by elevated dosage of MRPL36 and PET111. To further understand the gene expression defects caused by the cox2-22and cox2-27 mutations, we screened for nuclear genes that, when present in a high copy number, would suppress them. Thecox2-22 strain NB64 was transformed with a 2μm vector genomic library, and plasmids from transformants with increased growth on nonfermentable medium were isolated (see Materials and Methods). We further analyzed those plasmids which suppressed bothcox2-22 and cox2-27. These plasmids contained a gene encoding MrpL36p, which had been previously found to copurify with the large subunit of yeast mitochondrial ribosomes (28). A subclone containing only MRPL36 retained suppressor activity for cox2-22 and cox2-27, as well ascox2-22::ARG8m andcox2-27::ARG8m (Fig.4). Since the phenotype caused by the loss of MrpL36p had not been determined, we deleted the gene (see Materials and Methods). Cells bearing themrpL36Δ::URA3 allele failed to respire and became [rho −] and/or [rho 0]. This phenotype is typical of mutations that block all mitochondrial translation (34) and is consistent with the idea that MrpL36p could play a role in translation elongation as part of the large ribosomal subunit.
We also tested the ability of the COX2 mRNA-specific translational activator gene, PET111, to suppress the mutations in the leader peptide coding region. We found that elevated dosage of the wild-type PET111 gene (39), as well as a dominant allele that appears to have increased activity,PET111-20 (31), suppressed cox2-22, cox2-27, cox2-22::ARG8m , andcox2-27::ARG8m (Fig. 4). Taken together, these results are consistent with the idea that thecox2-22 and cox2-27 mutations cause similar translational defects that can be suppressed by improving the translation efficiency either at the level of initiation (PET111) or elongation (MRPL36).
Some intragenic suppressors of cox2-22 andcox2-27 cause translationally silent alterations in mRNA sequence that increase translation.Both thecox2-22 (NB64) and cox2-27 (NB140) mutant strains yielded spontaneous pseudorevertants exhibiting improved respiratory growth. Genetic analysis of 55 cox2-22pseudorevertants revealed that the slowest growing 23 contained as-yet-uncharacterized dominant nuclear suppressor mutations, while the most robust 32 contained mitochondrial mutations (see Materials and Methods). Eight of the independently isolated mitochondrial pseudorevertants were further analyzed by DNA sequence analysis of the 500-bp region of mtDNA surrounding theirCOX2 initiation codons. In parallel, 12 robust independent pseudorevertants of the cox2-27mutant were also analyzed by sequencing the same region of mtDNA. In each case, a single base substitution was identified in the coding region specifying the leader peptide or the first three amino acids of mature Cox2p (Fig. 5A). These intragenic suppressor mutations fell into three categories: (i) missense substitutions obtained only as suppressors of either cox2-22or cox2-27, (ii) missense substitutions obtained as suppressors of both original mutations (boxed in Fig. 5A), and (iii) base pair substitutions that do not alter the mutant pre-Cox2p amino acid sequence, obtained only as suppressors of eithercox2-22 or cox2-27 (circled in Fig. 5A).
Northern analysis of the pseudorevertant carrying thecox2-22, S8a allele, whose intragenic suppressor is identical to the cox2-27 suppressors S2 andS9, revealed no change in the steady-state level ofCOX2 mRNA relative to that of the wild type (Fig. 5B). A similar result was obtained for the translationally silentcox2-22 suppressor S3a (not shown). These results support the idea that suppression occurs at the translational level.
Western blot analyses carried out on the original pseudorevertants showed that Cox2p accumulation was restored to wild-type or nearly wild-type levels incox2-22,S8a and other pseudorevertants of bothcox2-22 and cox2-27 (Fig. 5B; data not shown). To monitor the effect of the suppressor mutations on reporter protein levels, cox2-22 was inserted with several of its intragenic suppressors into the cox2::ARG8m gene. Pseudorevertants were crossed with a strain (NB66) that harbors the cox2-60::ARG8m allele (6), a 130-bp deletion in the fusion construct that includes the leader peptide coding region. Reporter protein accumulation in the resulting diploid recombinants closely mirrored the strength of the original haploid suppressors on respiratory medium forcox2-22,S8a (Fig. 5B) and all other cases tested (data not shown). In addition, reporter protein accumulation was similarly increased over that directed by thecox2-27::ARG8m allele in recombinant haploid strains derived from the cox2-27pseudorevertants by cytoduction (not shown).
To confirm that the leader peptide mutations and their intragenic suppressors affected translation rates, we monitored relative rates of protein synthesis by pulse labeling cells in the presence of the cytoplasmic translation inhibitor cycloheximide and detecting mitochondrial translation products by autoradiography. The results confirmed that the rate of Arg8p synthesis directed by the mutantcox2-27::ARG8m mRNA was much lower than that of the wild-type reporter mRNA (Fig.6A). The rate of Arg8p synthesis was restored to various extents in the pseudorevertants (Fig.6A), largely consistent with the relative steady-state levels of Arg8p observed with the same strains (not shown).
To determine whether intragenic suppressor mutations would affect gene expression when placed in a normal context, we constructed haploid strains containing four suppressors of cox2-27 in otherwise wild-type COX2 andcox2::ARG8m genes. In no case did we observe a significant difference from the wild-type rate of synthesis of Arg8p (Fig. 6B) or Cox2p (not shown). Growth phenotypes and steady state levels of Arg8p and Cox2p were also wild type in strains bearing only the suppressor mutations (not shown).
The analysis of translation in the cox2-22 andcox2-27 mutants and their pseudorevertants suggests that both original mutations have similar negative effects on the rate of mRNA translation that are alleviated by intragenic suppressor mutations, some of which work on both mutations. In addition, they argue further that the nucleotide sequence of the mRNA encoding the leader peptide, rather than the peptide sequence itself, strongly influences the rates of COX2 translation.
Evidence that a stem structure in the COX2 mRNA coding region plays a role in regulating translation.Wild-typeCOX2 mRNA is predicted to form a relatively stable stem-loop structure extending from codon 10 through the first two bases of codon 17 (Fig.7A). Similar stems identical to the top part of the wild-type structure are also predicted for the cox2-27 mRNA, which lacks codons 2 to 6, and the compound allele cox2-22 (Fig. 7A). Interestingly, all of the cox2-27 intragenic suppressors and all but one of thecox2-22 intragenic suppressors (Fig. 5) affect base pairs in these predicted stems and would weaken them (Fig. 7A).
To test the possible significance of the stem, we constructed triple mutants that restored potential base pairing downstream of thecox2-27 mutation by compensating for several suppressor substitutions. We then examined the effects of the compensating mutations on the expression of thecox2::ARG8m reporter andCOX2. For example, a single predicted U:A base pair was disrupted by suppressors S12 (U to G) and S7 (A to C), each of which improved the expression of thecox2-27::ARG8m reporter (Fig. 7). However, when S12 and S7 were placed together in the same mRNA, generating a stronger predicted G:C base pair in the stem, reporter expression and respiratory growth were greatly decreased, to a level below that of cox2-27 alone (Fig. 7B). We observed similar decreases in reporter expression when we tested mutations that restored pairing to the predicted base pair disrupted by both cox2-27 suppressors S1 and S4(not shown). Disruption of this predicted base pair by combining theS1- and S4-compensating mutations in one mRNA increased reporter expression, as expected (not shown). Similarly, the addition of the S6 suppressor substitution to an mRNA containing S4 and the S4-compensating mutation also increased reporter expression, as expected (not shown). These data strongly support the in vivo existence of the predicted stem and indicate that its stability plays a role in controlling COX2mRNA translation.
Nucleotide sequence in the mRNA encoding the leader peptide is critical for COX2 translation, while the leader peptide amino acid sequence is not highly constrained.Several results described above suggest that elements in the leader peptide coding region, critical for translation in the context of downstreamCOX2 codons, are defined at the level of nucleotide sequence rather than amino acid sequence. To investigate this possibility further, we shifted the reading frame in this region to +1 by inserting a single nucleotide at upstream positions and deleting a single nucleotide at downstream positions in both the complete leader peptide coding region and a functional construct with deleted codons 7 to 14 (cox2-35) (Fig.8). These frameshifts caused wholesale changes in the sequence of the leader peptide, while minimally modifying the mRNA sequence. Surprisingly, all of these frameshift mutations, including cox2-39 (which lacks codons 7 to 14), behaved like the wild type for both respiratory growth and Arg+ prototrophy when placed in the COX2 andcox2::ARG8m genes, respectively (Fig. 8).
We conclude that the RNA sequence is clearly more important for translation in this context than the leader peptide amino acid sequence. Furthermore, membrane insertion, export of N-tail and C-tail domains and assembly of cytochrome oxidase can all be achieved with a pre-Cox2p leader peptide sequence that is very different from that of the wild type. However, it is important to note that in this context, the charge and hydrophobicity characteristics of the leader peptides generated by these +1 frameshifts are similar to those of the wild type. Thus, one could not exclude functional constraints on the leader peptide amino acid sequence on the basis of these data.
If mRNA sequence in the leader peptide region plays a greater functional role than the amino acid sequence, then multiple synonymous nucleotide changes in the region should affect gene expression. We made six such silent mutations in the first six codons of the functional construct with codons 7 to 14 (cox2-35) deleted to generate the cox2-41 mutation (Fig.9). In contrast to the corresponding frameshift mutation described above (cox2-39),cox2-41 abolished both respiratory growth and growth in the absence of arginine when placed in the COX2 andcox2::ARG8m genes, respectively (Fig. 9). This mutation had no effect on mRNA levels (Fig. 9B), but prevented accumulation of their protein products (Fig. 9C). Thus, changes in the cox2-41 mRNA sequence abolished translation despite the fact that this allele specifies a functional leader peptide amino acid sequence.
The leader peptide amino acid sequence appears to have a role in cytochrome oxidase assembly that is at least partially distinct from the translational role of the mRNA encoding it.We were unable to simply shift the leader peptide reading frame to −1 due to the occurrence of stop codons in that frame. However, we constructed a compound allele termed cox2-43, which was derived from the functional construct with a deletion of codons 7 to 14 (cox2-35), that was translated in the −1 frame: an upstream nucleotide was deleted, a downstream nucleotide was inserted, and two nucleotide substitutions were made to eliminate the resulting stop codons (Fig. 9A). These changes significantly altered the mRNA sequence and dramatically changed the character of the amino acid sequence.
This compound allele, cox2-43, had little or no effect on the level of COX2 mRNA (Fig. 9B), but prevented detectable Cox2p accumulation in cells grown at 28°C (Fig. 9C). Incox2-43 cells grown at 36°C, there was a low but detectable accumulation of Cox2p, possibly in its precursor form (Fig.9C), demonstrating that this mRNA is partially translatable. However, this mutation abolished respiratory growth at all three temperatures tested (Fig. 9A), suggesting that the mutant form of pre-Cox2p made in the cox2-43 mutant at 36°C is not functional. Interestingly, a strain bearing thecox2-43::ARG8m construct exhibited weak but significant growth on medium lacking arginine and contained easily detectable levels of Arg8p when grown at 36°C, confirming that this leader peptide mRNA sequence was partially competent in supporting translation at that temperature (Fig. 9A and C). Furthermore, the presence of PET111-20 on a high-copy-number plasmid greatly improved the growth of acox2-43::ARG8m strain in the absence of arginine at 36°C, but had no effect on the respiratory deficiency of a cox2-43 strain (not shown). In this context, it is important to note that it was previously found that low levels of Cox2p synthesis can be sufficient for detectable respiratory growth (31), while comparably low levels of Arg8p synthesis are insufficient for Arg+ growth (6).
Of all the mutations constructed in this study, cox2-43 is the only one that is competent for translation, albeit only at an elevated temperature, but encodes a nonfunctional form of pre-Cox2p. Thus, while the pre-Cox2p leader peptide amino acid sequence can be altered substantially without destroying function, it nevertheless appears to play a role in assembly of cytochrome oxidase at a step downstream of translation, and some amino acid sequences are incompatible with this role, at least at 36°C.
The cox2-43 mutant strain yielded spontaneous respiring pseudorevertants. We determined the mtDNA sequence in this region for three independent pseudorevertants that exhibited strong respiratory growth at all temperatures. All three contained identical deletions of the two codons immediately preceding the leader peptide cleavage site, as shown forcox2-43R1 (Fig. 9A). The cox2-43R1 allele supported wild-type respiratory growth (Fig. 9A) and wild-type levels of Cox2p (not shown). A strain bearing thecox2-43R1::ARG8m reporter construct grew well on medium lacking arginine at all temperatures (Fig. 9A). Thus, this two-codon deletion generated both an mRNA sequence exhibiting improved translation relative to cox2-43and a leader peptide amino acid sequence that supports cytochrome oxidase assembly.
Leader peptides on precursors of proteins inserted into membranes, or translocated through them, consist of amino acids sequences that generally play a role in protein targeting. However, the nucleotides encoding them are not typically involved in the regulation of precursor synthesis. In this study, we explored genetically the function of the 15-amino-acid pre-Cox2p leader peptide and the mRNA encoding it, specified by S. cerevisiae mtDNA. We examined the effects of mutations on the synthesis and function of pre-Cox2p and on the synthesis of a reporter protein, Arg8p, whose coding sequence was translationally fused to COX2 codon 91. Our data clearly demonstrate that the translation of the COX2 mRNA depends upon the mRNA sequence of codons specifying the pre-Cox2p leader peptide, in addition to its previously known dependence upon mRNA-specific translational activation by Pet111p through the 5′-UTL (17, 31).
First, we found that deletion of 13 leader peptide codons (2 to 14) from the cox2::ARG8m reporter mRNA prevented synthesis of the reporter protein despite normal mRNA levels. However, ARG8m was expressed from the COX2 locus in the complete absence of anyCOX2 codons. These findings strongly suggest that the first 91 codons of the pre-Cox2p coding sequence contain antagonistic elements that control translation positively and negatively: the positively acting element includes sequences in the first 14 codons specifying the leader peptide, while the negatively acting element appears to be within codons 15 to 91.
Our further analysis of mutations within the leader peptide coding sequence indicates that the positively acting element is complex or multipartite. None of the smaller deletion mutations we made in this region produced phenotypes as strongly negative as the deletion of codons 2 to 14 (cox2-20). Deletion of codons 2 to 6 (cox2-27) caused both leaky respiratory and leaky Arg− phenotypes without affecting mRNA levels, demonstrating the importance of this region for translation. However, residual growth of the cox2-27 andcox2-27::ARG8m strains indicates that the leader peptide codons remaining in this mutation, codons 7 to 14, also have a positive effect on translation in the absence of codons 2 to 6.
The positively acting element embedded in the leader peptide coding sequence functions at the level of nucleotide sequence, not amino acid sequence, since a shift of the reading frame through this region left the mRNA sequence relatively untouched while changing the amino acid sequence and did not detectably affect translation. On the other hand, when we altered the mRNA sequence of codons 2 to 6 (in the absence of codons 7 to 13) without changing the amino acid sequence they encoded (cox2-41), we completely eliminated translation. In this connection, it is noteworthy that the first six codons contain an 11-base sequence (AGAUUUAUUAA) that is also present one base upstream of the initiation codon in theCOX2 5′-UTL. It is tempting to speculate that the striking direct repeat of these bases plays a role in the positive regulatory function of the first six codons. The repeats could also play a role in the stringent selection of the COX2 initiation site, which they bracket. The ARG8m coding region lacks this sequence, yet is efficiently expressed in the absence ofCOX2 codons, indicating that repetition of this sequence in the coding region is not essential for general translation initiation or elongation. However, the sequence repeat could be important for antagonizing the negative element in downstreamCOX2 codons. A mutation altering the upstream copy of the sequence repeat in the 5′-UTL had a modest negative effect onCOX2 expression (11) and greatly reducedcox2::ARG8m expression (N. Bonnefoy, unpublished data), consistent with this possibility.
Two different but overlapping mutations in the leader peptide coding region behave similarly, suggesting that they affect the same mechanism. Translation of thecox2::ARG8m reporter was reduced to a similar degree both by deletion of codons 2 to 6 (cox2-27) and by a compound allele in which codon 6 was changed from AGA to CGU (both encoding R), and codons 7 to 10 (cox2-22) were deleted. Both of these alleles were suppressed by nucleotide substitutions clustered in the downstream part of the leader peptide coding region and, in the case ofcox2-22, the first three codons specifying mature Cox2p. Interestingly, we selected two different missense substitutions affecting codon 11 as suppressors of both cox2-27 andcox2-22, supporting the idea that the two original mutations impaired translation by a similar mechanism. In addition, some intragenic suppressors of each of the mutations were silent third-position substitutions in the COX2 coding sequence, consistent with our other data showing the importance of nucleotide sequence.
Similar stem-loop structures are predicted for the COX2mRNAs of the wild type, cox2-27, and cox2-22in the sequence corresponding to wild-type codons 10 through 17. This stem appears to exist in vivo and to play a role in reducing the translation of the mutant mRNAs, since all but one of the intragenic suppressors of these mutations that increased translation also weakened the stem. Furthermore, translation was reduced by compensating site-directed mutations that restored pairing withcox2-27 suppressor substitutions. Indeed, generation of a more stable stem, by conversion of a U:A base pair to G:C, reduced expression to a level below that of cox2-27. These results demonstrate that unfolding of the stem is necessary to improve translation of downstream coding sequences in the absence of codons 2 to 6. One possible interpretation of these data is that the residual positively acting sequences in codons 7 to 14 must be unfolded to function in the absence of codons 2 to 6. An alternative interpretation is that in the absence of codons 2 to 6, a stable stem could strengthen the action of the negative element downstream of codon 14 (this stem-loop cannot solely comprise the negative element since cox2-27 mRNAs are translated better than cox2-20 and cox2-41 mRNAs which lack it). These are not mutually exclusive possibilities, considering the potential dynamics of mRNA structure during translation.
We identified two nuclear genes, PET111 andMRPL36, as dosage-dependent suppressors ofcox2-27 and cox2-22. Pet111p is theCOX2 mRNA-specific translational activator (32,39, 51), a rate-limiting factor in COX2expression (17). Elevated Pet111p activity can also suppress mutations in both the mRNA 5′-UTL (31) and the initiation codon (6). Since a fraction of ribosomes are able to pass the leaky translational blocks caused by thecox2-27 and cox2-22 mutations, increased initiation would be expected to improve gene expression.
MrpL36p was previously found to be associated with mitochondrial ribosomal large subunits (28), suggesting that it may function during translation elongation. This protein, which we found to be essential for global mitochondrial gene expression, contains a central 80-residue region exhibiting recognizable similarity to the entire length of the bacterial L31 family of ribosomal proteins in a PSI-BLAST comparison. Little is known about L31, except that it is loosely associated with the large subunit of Escheridria coli ribosomes (12). Loose ribosomal association of MrpL36p could account for the unexpected isolation of a dosage-dependent suppressor encoding a component of the ribosome. Flanking the central L31-like region is a roughly 60-residue N-terminal sequence exhibiting no similarities to known proteins and a 60-residue C-terminal region with weak but nevertheless intriguing similarity in a PSI-BLAST analysis to a short region of E. coli Ffh, a protein subunit of the signal recognition particle. This region of similarity lies within the Ffh M domain, which binds to both the 4.5S RNA and the signal sequence of membrane proteins (16). Thus, one could speculate that MrpL36p might mediate regulatory interactions among the elongating ribosome, positive and negative elements in the COX2 mRNA coding sequence, and the nascent pre-Cox2p polypeptide to coordinate synthesis and translocation of the pre-Cox2p N-tail domain through the inner membrane.
The role of the pre-Cox2p leader peptide amino acid sequence in controlling synthesis and membrane insertion of the protein remains enigmatic. The leader peptide sequences from several fungal and plant species are not strongly conserved (20, 25), and animal forms of Cox2p lack it entirely. While a previous study indicated that the leader peptide causes membrane association of a passenger protein (21), our present findings demonstrate that several amino acid sequences, which were generated by frameshifts and various peptide lengths, can effectively carry out this and/or any other steps necessary for cytochrome oxidase assembly. For example, while the wild-type leader peptide is 15 residues long with an acidic group at the third position, we observed normal Cox2p accumulation and respiratory growth in a strain whose frameshifted leader peptide is 5 residues long, lacks an acidic group, and has a basic group at the second position (cox2-43R1).
Nevertheless, our genetic analysis indicates that the leader peptide amino acid sequence is not completely unconstrained with respect to function in cytochrome oxidase assembly. One allele,cox2-43, allowed synthesis of the reporter protein and Cox2p at reduced rates, but prevented Cox2p from assembling into active cytochrome oxidase. The leader peptide encoded by cox2-43 is strikingly different from the wild type and the other functional sequences we generated, in that it has positively and negatively charged residues (KD) just upstream of the processing site. Spontaneous pseudorevertants of cox2-43 all had identical deletions of the six bases encoding these charged residues, which allowed efficient cytochrome oxidase assembly and greatly improved translation of the reporter protein. It is striking that the pseudorevertant encoding an active form of pre-Cox2p also increased mRNA translation.
We propose that the positive and negative translational regulatory elements specified within the first 91 codons of COX2could function to ensure that continued translation of the mRNA occurs only if the nascent N terminus has successfully begun the process of membrane insertion leading to cytochrome oxidase assembly. While we cannot yet present a detailed model for how this feedback control mechanism might work, MrpL36p and Pet111p could function to convey information about the state of the nascent pre-Cox2p N terminus to the translating ribosome at the point where it encounters the negative element. We have also isolated dominant nuclear suppressors of both leader peptide mutations cox2-22 andcox2-27, which may identify other components of this system and lead to a better understanding of its mechanism. Passage through this element is likely to involve a dynamic interplay between alternative mRNA secondary structures and bound proteins.
Similar assembly feedback regulation of ATP synthase biogenesis could operate via pre-Atp6p, the only other yeast mitochondrial gene product with a leader peptide (30, 55). Such systems would resemble other translational feedback regulatory loops that couple the synthesis of specific components to the assembly of complexes. For example, translation of the chloroplast mRNA encoding cytochromef is coupled to assembly of the cytochromeb6/f complex in Chlamydomonas(9, 57). In Caulobacter, translation of the flagellin fljK mRNA is regulated by assembly of the basal body-hook structure (1). In this case, in which the flagellin is transported out of the cell by a type III secretion system, the regulation of fljK translation depends on sequences in both the fljK mRNA 5′-UTL and the first 9 codons of the structural gene. Finally, in the type III secretion system of Yersinia, signals necessary for both the translation and the secretion of Yop proteins have been mapped to the first 15 codons and shown to function at the level of nucleotide sequence rather than amino acid sequence (2). Thus, the regulatory system revealed by this study is likely to have its origins in the bacterial ancestors of mitochondria.
We are grateful to Geneviève Dujardin and Thomas L. Mason for supplying antibodies.
N.B. was a Human Frontier Science Program Organization long-term fellow (LT22/96) during the early stages of this work and is currently supported by the Association Française contre les Myopathies. This work has been supported by an NIH research grant (GM29362) to T.D.F.
- Received 8 September 2000.
- Returned for modification 16 October 2000.
- Accepted 19 January 2001.
- Copyright © 2001 American Society for Microbiology


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This pilot demonstration project is focused on building a resilient community capable of adaptation and implementing sustainable development solutions. Considering the community's needs, strengths, and aspirations, this project is primarily committed to addressing infrastructural frailties, reducing socioeconomic vulnerabilities, and elevating the living standards of Dhungentar locals in the long term, thereby building a resilient mountain village.
Importantly, the wide-ranging development and resilience-building activities of this pilot demonstration project will serve as a showcase for sustainable reconstruction and rehabilitation in a post-disaster situation, with a potential for up-scaling and wider knowledge dissemination.
The project was underpinned by four basic building blocks (comprising core activities) to reinforce its primary goals:

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text:
Punahou and the Worldwide Voyage – Converging Journeys
By Camila Chaudron '08
"The best indicator of our future is what we teach our children."
It was a late spring afternoon in 2013 when renowned navigator Nainoa Thompson '72 delivered this message aboard Hokule'a, sailing just a few miles off the beaches of Waikiki. With wind billowing in the iconic red crab-claw sails and the sun slowly setting beneath the horizon, the crew of the traditional voyaging canoe made its way east, toward Le'ahi (Diamond Head).
A group of Punahou teachers were on board that evening, connecting to Hokule'a during one of her last community sails before embarking on a 47,000 mile journey to circumnavigate the globe. That symbolic spring sail epitomized the ongoing partnership between Punahou and the Worldwide Voyage. Since then, the relationship between the School, its kumu (teachers) and haumana (students), and the values of the voyage have only deepened.
Shaping a Shared Vision
More than just a double hull and a deck, Hokule'a's mission is multifold and the depth of the canoe's promise is profound. Punahou has spent the past years developing programs that capitalize on the educational potential of the Worldwide Voyage through place- and inquiry-based lessons, interdisciplinary projects, extracurricular activities and professional development opportunities. The voyage has given students, teachers, parents and alumni the opportunity to support the School's mission to develop socially responsible citizens in a new and meaningful context.
That the Worldwide Voyage's mission is well-aligned with Punahou's priorities is another reason for synergy. Aptly named Malama Honua, or caring for the earth, the voyage ties into the global and local community's increasing focus on environmental sustainability. To understand the depth of purpose of the voyage, it's helpful to understand the etymological root of malama honua. "Lama means torch or light," explained Ke'alohi Reppun '99, a Hawaiian language teacher and social studies faculty at Punahou. "Malama comes from malamalama, which means to brighten or to illuminate."
Academy science students visited Hokule'a prior to the start of the Worldwide Voyage to meet with navigators and learn about traditional voyaging techniques.
Reppun interprets the leap from illumination to caring as follows: "When I care for you, I bring your needs and your well-being into focus. I illuminate you in my mind until I feel an aloha for you, and I begin to love and care for you. Malama is illuminating people and places so that we can bring attention and caring and balance to them." Honua likewise has many different connotations. Honua, Reppun explains, is any place where something can reside – it can exist in a kino (body), a classroom, a school, or extend broadly to the whole of planet Earth.
"Malama Honua provides an unreal opportunity to learn about new places because they've been illuminated by the voyage," Reppun said. "That's a mission I can stand behind, as the voyage makes each of us more knowledgeable about new places and people."
An Experiential Learning Huaka'i
Beginning in kindergarten, Punahou students discover how the lessons learned aboard the wa'a (canoe) can be applied to modern society. "We start with physical experiences for the younger children," explained Donna Reid '78 Hayes, a longtime kindergarten teacher. In the 2013 – 2014 academic year, every Junior School student and many Academy students visited Hokule'a and went aboard her sister canoe, Hikianalia, while they were docked near Sand Island. Once the wa'a embarked on their four-year journey in May 2014, faculty continued to provide opportunities for students to experience the magic of traditional Hawaiian sailing through local educational nonprofits, such as Kanehunamoku Voyaging Society.
"We asked students to reflect on needs versus wants aboard the canoe," Reid-Hayes said. On the wa'a, the importance of this question is amplified by the close living quarters, which require careful planning, cooperation and mutual trust. "The kids were able to steer the canoe and feel what it was like to voyage," Reid-Hayes said. They also learned about navigation – how the stars are used as trusted guides on the journey, and how the wind and the waves affect the wa'a.
These experiences inspired the children to bring the wa'a back to the classroom, where they decided to build a scale replica of Hokule'a. After they discussed the size and scale of the model, one parent volunteered to help with the construction of the hulls, another's grandmother and older sister helped teach the students to sew the sails and, after many months of building, they completed a model of Hokule'a.
"Everyone in the K – 1 Neighborhood works together," Reid-Hayes said, remarking on the project's success. The objective, after all, wasn't to create a model of the wa'a; Hokule'a is just the vehicle for bringing an experiential learning opportunity into the classroom in a hands-on, applied setting, driven by student inquiry and a values-based curriculum.
First-graders learned about Hokule'a's course to Tahiti from co-director of Gates Science Workshop and former crewmember, Tai Crouch.
Indeed, examples that incorporate these tenets seamlessly into the curriculum abound in diverse and age-appropriate ways across campus; third-graders learning about alternative energy sources within the context of ancient navigation; sixth-grade science experiments using wind speed and the size of the canoe sails as variables; eighth-grade outdoor education conservation activities tied to traditional cultural knowledge; ninth-grade social studies stewardship lessons at Pu'u o Manoa (Rocky Hill).
"Every day, I try to find new ways to connect the curriculum to the voyage and the values of malama honua," said Gail Peiterson, who reaches students K – 12 through the Gates Science Workshop and has been instrumental in developing the educational curriculum for the Worldwide Voyage.
Part of the beauty of these connections is that they're often interdisciplinary. Sixth-graders, Peiterson recounted, spend time working in the on-campus gardens to plant, grow and harvest foods that they later study, prepare and eat in the classroom. During Punahou's annual food drive, they connected the issue of hunger to the gardens by using Hokule'a as a metaphor for island earth. "When you have 30 crew members eating three meals a day, it adds up to 2,700 meals in one month," Peiterson noted.
"On the canoe it becomes visible, as does the trash produced," Peiterson continued. "Students understand that food is essential and this example illustrates how quickly the needs add up over time."
Educators as Malama Honua Ambassadors
Given Hokule'a's educational potential, many teachers across the state have adopted lessons from the Worldwide Voyage and made the mission their own. Some Punahou faculty, like Reid-Hayes, remember when Hokule'a was built and have taken the initiative to integrate these lessons into the curriculum because of their belief in the value of Hokule'a as a cultural ambassador of Hawai'i. "Connecting to the voyage helps students understand this unique place and keep the local culture alive," Reid-Hayes noted, "and there's a huge value in that."
Some, like Gail Peiterson and Tai Crouch, co-directors of the Gates Science Workshop, have always made environmental awareness a priority and the mission of malama honua provides an ideal device to integrate these lessons into the curriculum. In addition to Peiterson's educational partnership with the Worldwide Voyage, Crouch had served as a crewmember during Hokule'a's 1985 Voyage of Rediscovery. Yet, many Punahou teachers had no prior connection to Hokule'a's history or mission and the Worldwide Voyage provides a novel opportunity to learn and apply place-based lessons.
Faculty from various schools gather frequently throughout the year at Wa'a Talks to learn how their colleagues are integrating the lessons of the Worldwide Voyage into their curriculum.
Enter the Malama Kumu. An educational initiative spearheaded by K – 12 Hawaiian Studies Director Malia Ane '72, the Malama Kumu are representing Punahou locally and abroad as educational resources, cultural attachés and ambassadors of the malama honua mission. As Hokule'a makes her way around the globe, the Malama Kumu crew have met her at different port cities, organizing workshops and providing educational outreach. Already, they have traveled to Samoa, Aotearoa (New Zealand) and Australia, with further trips planned to South Africa and New York during the upcoming year. The faculty involved in the Malama Kumu initiatives represent a diverse cross-section of the School, from kindergarten art teachers to twelfth-grade science faculty.
"Visiting Aotearoa was a game-changer," said Eliza Leineweber '92 Lathrop, an Academy English and K – 12 garden resource teacher who manages several of the School's on-campus gardens. As the beginning of the Worldwide Voyage was oriented around the Pacific, "My involvement with the voyage brought me a deeper understanding of my own culture," Lathrop explained. This, in turn, informed Lathrop's relationship to Punahou's gardens, which are shaped by Hawaiian protocol, perspectives and culture. "I love experiences that remind me that, as an educator, I'm primarily a learner," she said.
"It's not just about the voyage, it's about bringing people together." – Malia Ane '72
The connections made through the Malama Kumu voyages have cut across country borders, created public and private school partnerships, and united the campus community. While the Malama Kumu initiative has so far allowed over a dozen Punahou teachers the opportunity to share and learn from communities abroad, the School is also committed to creating a local forum for educators to share resources, lessons and insights.
In partnership with the Hawai'i State Department of Education and Kamehameha Schools, Punahou helped launch Wa'a Talks: free professional development events open to Hawai'i teachers who want to integrate Hokule'a and the mission of malama honua into their curriculum. Wa'a Talks have brought specialists to the attention of educational institutions; connected teachers to crew members; and provided a platform for dedicated educators to share their materials, their successes and their challenges with a broader community. As Ane explained, "It's not just about the voyage, it's about bringing people together."
Students Take the Helm
"At Punahou, we have a shot at influencing kids who can really make changes," Ane reflected. "We want our kids to go out, make a difference and be leaders in different fields. If we can get them to love the reef, love the rainforest, their hearts will change their actions.
Punahou teachers understand the connection between empathy and action, and this insight is what drives them to create lessons that engage both the mind and the heart. The aim is for students to become self-sufficient, independently motivated learners so that their education continues beyond their school years. Every child is motivated by something different and, for some, Hokule'a could be the spark of inspiration for a budding engineer or a passionate ocean advocate.
Wreyn Waniya '18 became aware of the Worldwide Voyage after hearing about it in the news, and then through a Punahou field trip to Hokule'a. A few months after his class visited the docks, his wood shop teacher Stephen Wong reached out to Waniya to work on an extracurricular project: a student-teacher collaboration to design and build a model replica of Hokule'a.
"I'm grateful to Mr. Wong, who gave me this opportunity to make something by hand," Waniya remarked. The two worked together on the project for over a year, trading off responsibilities with the woodwork, tying the sails to the mast, adding support strings and applying the varnish as they attempted to make the model as authentic as possible. After a long and fruitful partnership, in the summer of 2015 they finished the wa'a and presented it to President Jim Scott '70 as a gift to the School.
"It's really cool that something I've worked on will be shared and displayed in public places," Waniya reflected. This project was so fulfilling that Waniya hopes to continue tinkering with his grandfather's woodworking tools. Meanwhile, the model wa'a will be used as an educational resource for Punahou students in years to come.
Wreyn Waniya '18 collaborated with his wood shop teacher, Stephen Wong, to create a scale model of Hokule'a that they gifted to the School for educational purposes.
Hokule'a's call can also transcend the classroom walls. Sarah Watanabe '15, for example, was inspired to deepen her connection to the movement in part because of the years she spent paddling outrigger canoes for Punahou under the guidance of Marion Lyman-Mersereau '70, former Hokule'a crew member and eighth-grade social studies teacher.
"Growing up, I always liked the ocean," Watanabe said, "and because of Ms. L-M, now I love everything about the ocean." In addition to homework and college applications, over the course of her senior year, Watanabe prepared to navigate around the Hawaiian Islands, learning about weather patterns and guiding stars. The summer after graduation, Watanabe participated in a 10-day sail around the Islands, steering her sailing school's vessel from Hawai'i island to Kaho'olawe.
"My kumu's kumu were some of the first navigators of Hokule'a," Watanabe said, explaining her relationship to the Worldwide Voyage. "They taught me that the oceans have never separated us, only connected us." Now Watanabe, who entered Boston University this fall, plans on continuing maritime activities in college and has already begun the training process to someday sail aboard Hokule'a.
"The hardest thing is coming back," she said. "I can't be selfish. I have to take what I've learned and share it with the community."
Inspired by her love of the ocean, Sarah Watanabe '15 learned about traditional navigational techniques and participated in a 10-day sail around the Hawaiian Islands.
Facing the Future: The Voyage Continues
"I love this school – not because I read about it in a book, but because I experienced it," said Ane, whose impact as a K – 12 administrator is as far-reaching as it is profound. "I want Punahou to continue to move forward. We need to tie lessons to experiences and we have the ability to provide out-of-the-box opportunities."
Faculty, including Ane, have already begun brainstorming new projects to connect Punahou students to the Worldwide Voyage in meaningful and relevant ways. Tai Crouch is in the process of applying for a grant to make a giant lau hala sail, which students and teachers could weave together.
"There's a lot that can be done with the sails," Crouch said. They require an understanding of mathematics in the planning stages, as well as artistry and cultural knowledge. Crouch has already found a source for the leaves, so if all goes according to plan, the handmade, full-sized canoe deck that teachers and students constructed last summer will soon have a set of sails to accompany it. "We need to continue to ask: How can we get more involved in this voyage, which has so much to offer?" Crouch said emphatically.
"I have to take what I've learned and share it with the community." – Sarah Watanabe '15
Third-grade teacher Denise Awaya '88 Wong has found that her young students relate best to the voyage when they can personalize their connections. Children in her class are encouraged to keep a journal where they create stories about the Worldwide Voyage by writing first-person narratives of themselves as crew members, which combines their knowledge of Hokule'a with their active imaginations. Wong's goal for this academic year is to integrate technology into the experience. "Students might pretend that they are at sea," she said as an example, "and act as crew members aboard Hokule'a using a green screen."
The driving motivator behind all of Wong's class projects is to instill the need to care for the environment. "I teach them that one person's actions affect others," she said. "What we do now is based on the past. And this plants the seed that we are all connected."
"The Worldwide Voyage is a common experience for the School and the state," Wong remarked. "When I was growing up, Hokule'a was a magical thing. It's powerful to know that somewhere in Hawai'i, people are teaching and learning from others because of Hokule'a."
Peiterson is also looking forward to exciting future projects. Punahou rented a giant map of the Pacific Ocean from The National Geographic Society that occupied the Gates Science Workshop last year, which allowed students to step into Hokule'a's sphere by tracing the route of the Worldwide Voyage with their feet. The rental was only temporary, so Peiterson has envisioned that students could create a scaled-down map of the world by outlining the continents on canvas. "Maps illustrate the relative distances between places," Peiterson said, "and underscore how large a percentage of our earth is covered with water."
Teachers acknowledge that the Worldwide Voyage is one of many important initiatives the School is addressing. "Hokule'a isn't the only thing happening right now," Peiterson said, noting: "There's the solar-powered airplane that landed in Hawai'i. There are ongoing ocean conservation projects happening worldwide. Hokule'a is one of many catalysts people can use to connect with the environment."
"To fulfill the mission of the voyage is to create relationships and connections to educational outlets," remarked Nainoa Thompson '72, president of the Polynesian Voyaging Society and a visionary in the field. "It takes progressive, fearless institutions that understand the right thing to do in preparing children for tomorrow and have the courage to do it – a place like Punahou," Thompson said during the springtime faculty voyage aboard Hokule'a.
"In a broad way, what happens on this canoe is like a school – it's a very powerful and intense learning platform," Thompson said. "It's important to have these teachers on board because they're the ones that create the exponential reach we need to change the world."
About the Worldwide Voyage
What: Circumnavigation of the globe using only traditional navigation techniques aboard the Polynesian voyaging canoe Hokule'a, meaning star of gladness.
Who: Master navigators, apprentices, documentarians, scientists and educators are all actively participating on the voyage, while students can follow its progress at home and in the classroom at www.hokulea.com.
When: Malama Honua is a four-year journey that began in May 2014, with a planned return date in 2017. Prior to Hokule'a's launch, she also traveled around the Hawaiian Islands in 2013 to spread awareness about the voyage and its mission.
Where: Hokule'a and Hikianalia (her sister ship) began their voyage together, launching from Kualoa on O'ahu and traveling together around Polynesia before reaching Aotearoa in late 2014. From there, Hikianalia returned home to the Hawaiian Islands while Hokule'a continues her journey westward, traveling around Australia, through Indonesia and eventually reaching the southern tip of Africa later this year. The plan for the next two years is that she will also visit South America, North America and Europe, returning to Hawai'i through the Panama Canal.
Connecting Punahou Alumni to the Worldwide Voyage
"Many people on the East Coast view Hawai'i as just a tourist destination; there's a lot of education needed to share the values and the culture of Pacific Islanders here," said Leslie Morioka '64 during a phone interview from New York City.
Morioka sits on the board of directors of Halawai, a New York-based nonprofit organization whose mission is to build appreciation and understanding of Pacific Island cultures. Halawai is leading the charge to organize events around Hokule'a's visit to the city in June 2016, which will hopefully intersect with the United Nations World Oceans Day.
"Hokule'a is just the beginning," Morioka noted. The hope is that the Worldwide Voyage will activate a global community to connect people of all ages with the mission of malama honua. Halawai is actively partnering with public and independent schools at all levels in the New York area, from pre-K to grade 12.
"There are 1.1 million students in New York City, and if we share the values of Hawai'i – respect for island earth, the ocean – they can extend these principles to themselves and their lives," Morioka explained. "We look forward to working collaboratively with the Punahou Alumni Association New York chapter, as well as other local organizations to build programming so that every day, we're doing something with regards to the environment."
If you live in New York and would like to volunteer, email email@example.com.
Hokule'a's Historic Voyage
When Hokule'a made her maiden voyage to Tahiti in 1976, alumni were heavily involved in the endeavor – in fact, Punahou had the largest total representation on board of any school. "It was the year of Hokule'a," the fall 1976 Punahou Bulletin proclaimed in a two-page feature about life aboard the traditional canoe. Tommy Holmes '63, Dave Lyman '61, Kimo Lyman '68, Keani Reiner '70 (one of only two women selected to be part of the crew) and Nainoa Thompson '72 all participated in the initial voyage. The legacy of that historic voyage is still alive at Punahou, particularly because of the strong family ties. The Lymans' younger sister, eighth-grade social studies teacher Marion Lyman-Mersereau '70, is a former crewmember. Her son, Junior School PE teacher Kaniela Lyman-Mersereau '05, has now sailed on several legs of the Worldwide Voyage in the Pacific, off the coast of Australia all the way to Bali, Indonesia.


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- God created a universe to display the glory of his grace.
- So all should act in a way that draws attention to this glory.
- The obedience and service of God's people is most glorifying when they are most dependent on his grace.
- Praying for God's help is perhaps the key way God preserves and maintains the dependence of his people on this grace and power.
- When the Spirit prompts and directs our prayer, the ultimate purpose of the universe is therefore fulfilled. God is glorified for his grace: the Father for initiating his reconciling work, the Son for purchasing it, and the Spirit for leading us to benefit from it.
Thursday, 23 September 2010
- Theology
- Posted By: Jon Hobbs


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Located at the Greenland Urumqi Twin Tower of 258 meters high, WEPLUS Urumqi Greenland Center Space lies in the heart of Hongguang Mountain international exhibition zone. The Twin Tower with 40,000㎡ coverage features three Grade A office buildings which lead the new economic era of Urumqi.
Landmark: Exhibition Center; Urumqi Culture Center; Olympic Center; Hilton Hotel, Hongguang Mountain Park
Entertainment: Hongguang Mountain 5A scenic spot; Happy Valley (under construction) ; Mustang Silicon Valley (under construction)


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Browse by Uncontrolled Keywords
Group by: Year | Authors/Creators | Item Type | Full Text
Jump to: 2002
Number of items: 1.
Morgan, D; Mahe, C; Mayanja, B; Okongo, JM; Lubega, R; Whitworth, JA; (2002) HIV-1 infection in rural Africa: is there a difference in median time to AIDS and survival compared with that in industrialized countries? AIDS (London, England), 16 (4). pp. 597-603. ISSN 0269-9370 DOI: https://doi.org/10.1097/00002030-200203080-00011 Full text not available from this repository.


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I am trying to set up a network of sites (a subdirectory based multisite) which I want to be available on Spanish & English. Each site will work as a sub-brand of the main brand (the main site)
I am wondering if it would be possible to build an structure as this one:
mybrand.com/es (Spanish versión of site 1 (mainsite))
mybrand.com/en (English versión of site 1 (mainsite))
mybrand.com/subbrand1/es (Spanish versión of site 2)
mybrand.com/subbrand1/en (English versión of site 2)
mybrand.com/subbrand2/es (Spanish versión of site 3)
mybrand.com/subbrand2/en (Spanish versión of site 3)
Is that possible? how can I create subdirectories based sites within sites of a multisite without having to reinstall wordpress on each of them? it means creating a multisite nested on a multisite, isn't it?
Thank you


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Boob physics on your OLED screen… or a nice fighter you might enjoy.
Team Ninja lead designer Yosuke Hayashi has announced that a port of the 3D fighter Dead or Alive 5, called Dead or Alive 5 Plus, will be arriving on the PlayStation Vita early next year. Not much information was supplied, but the title of the game suggests that it will feature extra content.
The Vita already has a strong library of fighting games (UMvC3, Mortal Kombat, SF X Tekken, BlazBlue: CSEX) and with this new addition to this list, the PS Vita must surely be a purchase worthy considering for a gamer with a passion for fighting games in combination with portability.
The announcement:


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The Buys Family: A Family Session in Rexburg Idaho
So, let me state that Anna's family is actually perfect in real life, so this is a great example of perfect family photos.
Anna is not only a great friend of mine, but a talented photographer who shot my birth and maternity photos as well. I was honored to capture her family's session!
I had to share their story all over the internet, so here it is!
Tell me about your kids! How old are they?
Milo is 3 and Oliver is 1.
How did you and your husband meet?
Kent and I met in Rexburg Idaho at BYU-Idaho in the Ricks building. I went up to him and said "hey, I think you were in my math class." He didn't remember but we started hanging out and here we are now!
What do you love most about Rexburg?
I love that Rexburg is a smaller town with so much growth and potential to meet new people every day! We love how the college is here and this is where our life started together. We graduated here, our boys were born here, built my photo business here and we are grateful to call it home.
What's the best part about being a mom?
The best part about being a mother is watching them grow and learn new things. They bring me the most joy and I'm so grateful to be their mother. I'm learning and growing right there with them.
How do you choose outfits?
When choosing outfits I try to stick to neutrals so that the attention stays on our faces.
How do you choose a photographer that's right for you?
When choosing a family photographer it's important that they are patient and happy around kids! Celine was amazing!!
What are your favorite things to do as a family?
We love to play chase in our house and eat teriyaki chicken at "the panda store" (panda express). We also love to dream as a family and talk about all the dreams Milo has (fly an airplane, drive an excavator and a combine harvester tractor)!
Loved being able to work with this amazing woman!
Here are some highlights from their gallery:


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Have you seen Margot Robbie as Queen Elizabeth I? Probably not, because she's seemingly unrecognisable.
Pictures have emerged of the 27-year-old wearing a thin, balding red wig and an outrageous amount of foundation.
The Aussie actress stars as the former monarch in upcoming movie Mary Queen of Scots.
The biopic stars David Tennant, Downton Abbey's Brendan Coyle, Dunkirk's Jack Lowden, Martin Compston, Joe Alwyn, Guy Pearce and Saoirse Ronan.
i refuse to believe that this is margot robbie pic.twitter.com/RJOmb2SyTV
— lu (@pcrtmans) August 20, 2017
Margot isn't the first actress to tackle the role.
She follows in the footsteps of Judie Dench, Bette Davis, Helen Mirren, Cate Blanchett and Miranda Richardson.
Margot Robbie in London filming "Mary Queen Of Scots" (August 2017) [1/3] pic.twitter.com/5n38aTf0Hk
— Margot Robbie Daily (@MargotRobbieRus) August 21, 2017
The film will tell the true story of Mary Stuart's attempt to overthrow her cousin Elizabeth from the throne.
It's quite a departure from her role as Harley Quinn, which she will be reprising alongside Jared Leto in an upcoming Joker-Quinn movie.
Margot's latest release I, Tonya premiered yesterday at the Toronto Film Festival to positive reviews.


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For the USC and Exposition Park neighborhoods in Los Angeles
Presented by Dwight Trible, Executive Director, The 'new' World STAGE Art, Education & Performance Gallery non-profit 501(c)(3) "BEST South L.A. Jazz Club" - LA Weekly
Fri., 6th - Sheri Brown
Sat., 7th - Acoustic Revelation Band
Views: 3310
L.A. previews March 22-28: ..., Karen Briggs, Charles Owens, ...
Fri. March 22 -- Violinist Karen Briggs stretches your melodic-(J)azz expectations w/ bassist Robin Bramlett, drummer Forrest Robinson & keyboardist Nick Smith. $25.
Sat. March 23 -- W/ the chops of a studio musician & the imagination of a Picasso, South L.A. sax mainstay Charles Owens leads a band topmost in feel & experience featuring pianist Theo Saunders, bassist Henry Franklin & drummer Don Littleton. $20.
(both @) The World Stage, 4321 Degnan Blvd., Leimert Park (Village) 90008; 9pm (two sets); www.theworldstage.org.
BRENDA LEE EAGER "Women's Her♀Story Month...The World Stage
Los Angeles' Leimert Park, CA
MAIA "Women's Her♀Story Month" •...The World Stage
Los Angeles' Leimert Park, CA
Wuz Hot? Weekly Best Bets 4 Live Jazz L.A.
losangeles.jazznearyou.com: LOCAL CALENDAR - Upcoming / Just Added
JAMES H. LEARY Legacy Ensemble • DCA Compose LA...The World Stage
Los Angeles' Leimert Park, CA
ANTHONY WILSON ● DwighTrible Presents "...The World Stage
Los Angeles' Leimert Park, CA
"Jazz Near You, about local concerts and events."
L.A. previews February 15-21: Doug Carn, Dwight Trible,...
Fri. Feb. 15 -- '70s Black Jazz Records organist Doug Carn forms a topline trio with saxist Teodross Avery and drummer Marvin "Smitty" Smith. At the World Stage, 4321 Degnan Blvd., Leimert Park 90008; 9pm (two sets); $25; www.theworldstage.org.
Tues. Feb. 19 -- Cosmic Vibrations is the superlative ascensional ensemble of singer Dwight Trible, bassist John B. Williams, windman Pablo Calogero, percussionist Derf Reklaw, multi-instrumentalist Christopher Garcia and drummer Breeze Smith. Highly recommended if you're in the nabe. At UCLA's Jan Popper Theater, 445 Charles E. Young Drive, Westwood 90095; talk 7:30pm, concert 8pm; FREE; register here.
KKJZ Community Calendar Feb. 2019
Presented by Dwight Trible, Executive Director, The 'new' World STAGE Art, Education & Performance Gallery non-profit 501(c)(3)
FEBRUARY (workin'-up2date *updatez*) From: the-desk-of DT**
Fri., 1st - Dexter Gordon Tribute w/ Justo Almario & Teo(dross) Avery + Eric Reed, Mike Gurrola & Anthony Fung 8pm *$20*
Sat., 2nd - Joshua White - Alex Cline Duo 9pm *$20*
*Thu., 7th - Kahil El'Zabar, Ethnic Heritage Ensemble 9PM $25** (Event - date, time & program changed)**
Fri., 8th - Willie Jones III 9pm *$20*
*Sat., 9th - JuiniI Booth / Robert J. Carmack, "Black as a 1000 Nights" The Artistry & Poetry of Sum Ra - National Black History Event 9pm *$25* (Event - headliners & program changed)**
Fri., 15th - Doug Carn w/ Dr. Teodross Avery, Marvin "Smitty" Smith... 9pm *$25*
Sat., 16th - Ladies Love Laughter 9pm *$10*
*Sun., 17th - James H. Leary Legacy Ensemble 3pm (free) RSVP (date & time mix-up w/ 3/17 Event, unconfirmed)*
Fri., 22nd - Father Amde (Watts Prophets) 9pm $20
Sat., 23rd - Anthony Wilson 9pm $20
foot ♪ - ¿hav been unable 2 post ₵ Jan. '19?
A kind, RISE music-segue-mentionin'; Thu 7th - Kahil El'Zabar, Fri. 8th - Willie Jones lll & Sat. 9th - Juini Booth
Live On-Air shout-outz by our "Humble Facilitator" Host - Mark Maxwell (20 yrs.)
Sun.-nite, 12am midnight until Mon. 3am
"a weekly creative (J)azz celebration of African roots, social consciousness & spiritual transcendence". Stylistically it generally revolves around a specific sub-genre that has come to be called "spiritual (J)azz"; however, I often touch base w/ many other facets of the (J)azz tradition. It is mostly a music mix show w/ occasional live performances & interviews as well as regular announcements regarding local community events & performances. - /\/\/\/\


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Colorado in the fall is magical. The light, the colors, the warm days and cool evenings. It's the perfect time of year to shoot in. So it's really no surprise when all of my Colorado families want to shoot in the fall!
This past year, I decided to host a mini session marathon for the Performance Ready Fitness Studio members, and I was so happy with everyone that participated! I love these ladies and their families like my own, and we couldn't have asked for a better day!


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Google Participate in Audiobooks is getting a important update currently that adds a selection of new functions to the company that were being sorely lacking when it released earlier this 12 months. None of these are groundbreaking, but they'll enable Google attain element parity with some of its competition even though injecting a little bit of its proprietary smarts into the procedure, also.
Maybe the most helpful new element in today's release is Sensible Resume. Rather of finding up in the center of a sentence or even term when your audiobook playback receives interrupted (maybe by Google Maps supplying you instructions or a welcoming passerby who is asking for instructions even though you are evidently listening to an audiobook). Dependent on the duration of the interruption, this new element will neatly rewind to the starting of the term or sentence to enable you remain in the circulation.
Also new in this update are the skill to established bookmarks so you can quickly go back to your favorite section of a reserve and the skill to velocity up the audio — or slow it down so you can definitely savor your favorite passage in Ulysses. Both of those of these functions were being unquestionably lacking in the initially release.
If you're a regular Google Assistant person and are already earning use of the not long ago released Routines element, you'll be happy to hear that you can now opt for to continue on your audiobooks when you wake up or start your commute.
And if you have household which is spread all around the earth, you'll be happy to hear that assistance for Google's Family members Library, which lets you to share Google Participate in purchases like applications, online games, movies, e-textbooks and audiobooks, is now rolling out in 13 new countries: Belgium, Germany, Italy, Netherlands, Norway, Poland, Russia, Spain, Switzerland, Chile, Mexico, Japan (audiobooks only) and South Africa.
All of these new functions are now accessible on iOS and Android.


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Crystal De La Rosa
Originally from San Antonio, TX, Crystal moved to Austin in 2006 where she obtained a business degree consulting and change management from the University of Texas at Austin. Upon graduation she join...View Profile
Sage Wilson Property Group is a client-focused team of experienced Realtors® with over 30 years of combined real estate experience. We are Austin experts! Our growing team is here to help you make the best possible real estate decision, whether you're new to the area, selling your home or looking to invest in Austin's booming real estate market.
There are many qualities and skills that go into being an excellent real estate professional – integrity, in-depth community and market knowledge, marketing savvy, effective negotiation skills and a high-quality professional network, all of which are hallmarks of how we work. At Sage Wilson Property Group we leverage over thirty years of sales, marketing, project management and customer service experience for the benefit of our clients.
We believe that providing the very best service is about putting our clients first. This means being accessible, being a good listener as well as a good communicator, and responding quickly to your needs.
This "client-first" philosophy has always been our approach and it requires us to continually improve our skills and ways of doing business.
We take deep satisfaction from helping people with their real estate needs, and will provide you with personalized, high-quality service that will make your experience of buying or selling as enjoyable as possible.
Originally from San Antonio, TX, Crystal moved to Austin in 2006 where she obtained a business degree consulting and change management from the University of Texas at Austin. Upon graduation she join...View Profile
April Flores, was born and raised in Texas, and has been a resident in Central Texas since 2004 with 1 year interrupted in Florida. She received a Bachelor's Degree in Economics and Master's Degre...View Profile
Originally from Michigan, Stacey made her to move Austin Texas in 2011. She graduated from Michigan State University with a B.A. and minored in Graphic Design, Photography, and Ceramics. Stacey has an...View Profile
Amy made her move to Austin in 2006 after a successful real estate career in Chicago that began in 1990. Amy brings a wealth of experience from her years as a legal assistant to a real estate attorney...View Profile
Erik is a licensed broker in the State of Texas and has lived in Austin since 1997. He can give you insight into investment opportunities and market trends in the commercial and luxury residential re...View Profile


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Bristow Academy, located in Titusville Florida, is introducing a wildland firefighting course for current and prospective helicopter pilots. This course, entitled Wildland Fire Orientation for Rotary Wing Pilots, is a ground school designed to provide pilots with basic knowledge of wildland fire behavior, firefighting safety and tactics and the use of helicopter in fire suppression and prescribed burning operations.
SAN DIEGO, CA December 7, 2011 – Component Control announced today Flight Power Repair Group, an FAA / EASA certified repair facility for the Pratt & Whitney JT8D engine line, has selected Quantum Control to manage its specialized repair facility in Doral, Florida, whose services include the overhaul of Disks (LLP's), Spacers, Stators, Cases, Housings, Tubes and Ducts. Flight Power Repair Group is leveraging Quantum to automate all repair shop and materials management operations, ensuring best practices across their business.
We need help today in raising money to fund our scholarships. Currently, our largest scholarship being offered is $10,000. Sure, we could offer smaller scholarships that only cover a portion of training so that we would be able to offer more scholarships...but that's not what The Doug Doty Vertical Flight Foundation is about.
During LIMA (Langkawi International Maritime & Aerospace Exhibition) in Malaysia, Turbomeca (Safran group) announce that its Arriel and Makila engines, powering 21 helicopters of Malaysian Helicopter Services (MHS), clock up today one Million flight hours. MHS is today the largest civil helicopter operator in Malaysia.
Thousands of helicopter professionals from 160+ countries "worldwide" visit JustHelicopters.com every day, making it the Helicopter Industry's #1 Online Resource! Whether a Helicopter Pilot, Helicopter Student, Helicopter Mechanic, Employer, Helicopter Flight School, Helicopter Business, or an enthusiast, JustHelicopters.com has something for you.
© Copyright 2000–2012 Justhelicopters.com
Terms Of Use


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There are several companies who work with their customers to find the antique car parts they need. They are much like a locator service. You request a part and they put the leg work in to find out where the part can be purchased from. You then pay them a finder's fee and purchase the particular antique car part from the source they quoted.
Start to loosen the wheel lugs. When lugs are loosened sufficiently (not all the way), jack up the front end of your vehicle and rest it on jack stands. This will make it safer to work under your vehicle as opposed to using the jack alone.
PPG reported strong first-quarter results on April 15. It had sales of .1 billion, up 12% versus the prior year's first quarter. PPG had earnings of .70 per share, topping the Zacks Consensus by 7 cents, or 11.1%. For the last five quarters, PPG has beaten the Zacks Consensus by an average of 22.0%.
Another mistake people make is they do not choose a market that can be profitable for them. The best way to make money with affiliate marketing is to identify a problem, determine that people are seeking ways to solve that problem, and then help them do it.
Buying discount from salvage dealers can save you some in the long run. Why buy new if your auto is older and may be sold in the near future. Some places are called pick a part because they allow you to visit and search there yard for what you need however you will have to take it off.


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Dear Neil: I have been in an on again-off again relationship with this beautiful lady since this last Christmas. She and I are close when we are together, and her kids absolutely adore me. But she was in an abusive relationship with the kids' father. Now when she and I are close, she finds the very next thing I do that is unacceptable to her and she uses it as an excuse to run away. She will end all communication for a couple of weeks, and then we will get back together again. I love her and really want her to give me an honest shot, but so far she's been unable to do so. How do I help her to get over her past and not run away?
On Again-Off Again in Portland
Dear On Again: You could start by keeping your perspective. You are in a two-month relationship with a woman who has an intimidating past, and you want her to be available for a relationship with you right now—with no hesitations or reservations. You are not looking at whether she is ready to be in a committed relationship, whether she has made peace with the ending of her previous relationship, if she thinks it is wise to let her kids get close to you this early on, and you aren't sure that she knows how she really feels about you.
Consider the possibility that you may be expecting her to move too fast and to be ready for a relationship too quickly. It's entirely possible that you are her rebound relationship (which would assist her in feeling "normal" again. She may not be ready for a committed future with anyone yet.) The getting-to-know-you process takes some people more time than it does others, and you may simply be more ready for a relationship than she is at this time.
However, let's say you are right, and that she is pushing away from you because she has low trust toward men, or is leery of getting close to a man quickly, or she hasn't gotten over the trauma from her previous relationship. You could talk with her about this subject, asking her what she would need from you in order to not push away—and what she is attempting to communicate to you when she pushes away. The bottom line is that you cannot help her get over her past, but you can demonstrate your compassion and trustworthiness over and over again. Hopefully she will begin to feel increasing levels of safety and trust around you.
Tagged: getting over the past how do I stop my girlfriend from withdrawing?
Has your relationship turned cold and distant? Neil's book Love, Sex, and Staying Warm can help you rekindle your passion.Get My Book Today →
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Sony Xplod Car CD Receiver Price in Pakistan | CD Receiver for Cars
- Sony Xplod Car CD Receiver Price in Pakistan | CD Receiver for Cars
- Company : Sony
- Product Type : CD Receiver
- Price In Pakistan : Rs. 7,650 To Rs. Rs. 13,600
- Prices
- Sony WX-GT77UI Price13,600 (Pak Rupee)
- Sony CDX-GT700UI Price9,800 (Pak Rupee)
- Sony CDX-GT600UI Price8,200 (Pak Rupee)
- Sony CDX-GT500U Price8,000 (Pak Rupee)
- Sony CDX-GT490US Price7,650 (Pak Rupee)
One of the CD receivers of Sony is WX-GT77UI. Price is a little high but it has some distinguishing features which make it unique. So it has one USB port through that one can connect an iPod or phone from that receiver to listen to their favorite music. Sony CD receiver also has a 13 segment LCD for display. It has quick browser in it; one can easily find music of their choice. There is a radio option in it, so one can easily hear anything played live. This device also allows to listen to music in CD or MP3 in their original vibrancy. There is an inbuilt subwoofer to improve the quality if the sound. Though, this receiver is of 52 Watts, however loud and powerful enough to be heard from a distance. The color scheme can be changed to blue or green. Even it has a wireless card remote. The car will be in the musical air while using this receiver.
Another Sony CD receiver CDX-GT700UI, which caters to the needs of moderate multimedia enthusiasts. The basic features of this CD receiver are that it has a USB input into it. You can even listen to radio or MP3 CDs. The face plate can be flipped down and it is removable. Some other features are quite common like rotating volume controller, wireless remote controller, 3 pair RCA output.
Sony has one other receiver that is CDX-GT600UI, which slightly lower in price than the previous one. It has many features in it like all other CD receivers have. The FM tuner in it can be turned off if you don't want to listen. It also supports USB port for connecting iPod into it. The LCD display is the same as the other versions have. It has quick browser but it has limited features as compared to other versions which are discussed above.
The other model Sony CD receiver is being CDX-GT 500U, its price is even lower than the previous model. The features are just according to the price tag it bears. It has same features like LCD display, FM Tuner is present, and MP3 is even there. The USB port is their so almost same thing but price is cheaper.
Sony even has a car CD receiver model which is named as CDX-GT490US and the price is really cheap just for being below Rs. 8000. Features are same as other model have but with the cheaper price it has a LCD, FM tuner is in it. USB port is present even the quick browser is in it.
The prices in Pakistan are the one which are listed above. The performance of the receivers is amazing but every model has different features according to the price range.
- LCD Panel Size 7″ and 8″
- Internal Memory 128MB, and 2GB
- Still Image Playback
- Lightweight for Maximum Comfort
- Slideshow Playback
- USB Mini B-type (Hi-Speed)
- Clock and Calendar
Edifier Speakers Price in Pakistan
Model M1350 Prices Rs. 2,500 (Pak Rupee) Edifier Multimedia Speakers M1350 Features 2.1 multimedia speaker system 3.5mm Stereo Line-in Flat panel style satellites Built-in headphone jack Unique down-firing subwoofer 6.2″x8.9″x8.2″ (WxHxD) Subwoofer 2.7″x6.5″x3.4″ (WxHxD) Satellite 2.8 Kg Weight Model R401 Prices Rs. 4,500 (Pak Rupee) Edifier Multimedia R401 Features 2.1 multimedia speaker system 3 inch […]
Apple iPod Touch Price in Pakistan
The Apple iPod touch is a revolutionary innovation by Apple which is slim, light and classy beyond your dreams! This iPod has so much to offer, from a great camera to thousands of Apple apps. Now interact with your friends on Facebook or listen to music all day long with this amazing iPod. Here is a review…
LG Car Audio Price in Pakistan
The new LG car audio is Slot-In type with an appealing and stylish display, front Audio AUX In, Illumination and Dual Display. The player is capable of playing the media through USB, CD plus it is has Tuner with FM/AM. The Quick Jump is placed to simply and quickly search and play your song.
LG DVD Prices in Pakistan | LG Video Prices
The DVD player model no BD 660 from LG has some unique feature and is a blue ray player. Blue ray is the new format of an optical disc that allows massive storage and is developed by electronics companies like apple, Samsung and Sony in collaboration with blue ray disc association.
LG Home Theater System Price in Pakistan
LG has a variety of wide range of home theaters. LG home theaters are known for its quality. One can enjoy listening songs and watch movies through them. In 2012 LG launched a huge range of home theater systems worldwide. This system can enhance the beauty of the home.
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