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Genetics, Vol. 169, 1415-1424, March 2005, Copyright © 2005
doi:10.1534/genetics.104.027615
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Department of Plant Biology, University of Minnesota, Saint Paul, Minnesota 55108
1 Corresponding author: Department of Plant Biology, University of Minnesota, 250 Biological Sciences Center, 1445 Gortner Ave., St. Paul, MN 55108.
E-mail: pete{at}biosci.cbs.umn.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Flagellar length in the unicellular, biflagellate, green alga Chlamydomonas reinhardtii is tightly regulated. Wild-type cells display a narrow distribution of flagellar lengths between 10 and 15 µm, never exceeding 16 µm. Mutants with abnormal flagellar length include both long-flagella (lf) mutants, with flagella up to three times the length of wild-type cells, and short-flagella (shf) mutants, with flagella approximately one-half the length of wild-type cells (MCVITTIE 1972a; JARVIK et al. 1984; KUCHKA and JARVIK 1987; BARSEL et al. 1988; ASLESON and LEFEBVRE 1998).
Chlamydomonas cells actively maintain two flagella of equal length. Within 2 hr after amputation, wild-type cells regrow their flagella to predeflagellation lengths. If one of the two flagella is severed, the remaining flagellum immediately begins to shorten while the amputated flagellum begins to regrow (ROSENBAUM et al. 1969). This simultaneous shortening and lengthening of the flagella continues until the two flagella reach the same length; the two flagella then grow out together to the wild-type length. The control of flagellar equality is further demonstrated in the null mutants of LF3 that have an unequal-length-flagella (Ulf) phenotype; these mutants are mostly flagella-less, but under certain growth conditions, two flagella of unequal length are produced (TAM and LEFEBVRE 1993; TAM et al. 2003). The null lf3 mutants have lost the ability to enforce the equality of length between the two flagella.
The most striking demonstration of the dynamic nature of flagellar length control is the behavior of lf or shf mutants during mating. Chlamydomonas gametes fuse to form a temporary dikaryon cell with four flagella. When a wild-type cell is mated to an shf mutant, the two short flagella rapidly elongate to wild-type length (KUCHKA and JARVIK 1987). When a wild-type cell is mated to an lf mutant, the two long flagella shorten to wild-type length within 15 min (STARLING and RANDALL 1971). Even though 20–40 µm of flagellar material is resorbed into the dikaryon cell in this experiment, the wild-type flagella do not lengthen, implying that the growth of flagella is actively limited rather than being a passive response to the pool size of flagellar proteins. These observations demonstrate that a mechanism contributed by the cytoplasm of the wild-type gametic cell recognizes abnormal-length flagella and restores length control.
In Chlamydomonas, four long-flagella genes (LF1, LF2, LF3, and LF4) have been identified that can be mutated to produce cells with abnormally long flagella (MCVITTIE 1972a; BARSEL et al. 1988; ASLESON and LEFEBVRE 1998). Within a population of lf cells, the flagellar length distribution is broad, and the flagella can reach lengths three times that of wild-type cells. Some lf mutants, including lf1 and three of the five alleles of lf2, also demonstrate a severe defect in regenerating flagella. These strains do not fully regenerate their flagella up to 24 hr after amputation (MCVITTIE 1972a; BARSEL et al. 1988). Genetic analysis of double and triple mutants of LF1, LF2, and LF3 suggests that these genes lie in the same regulatory pathway. The double mutants, lf1 lf2, lf2 lf3, and lf1 lf3, exhibit a synthetic, flagella-less phenotype (BARSEL et al. 1988). This phenotype is dependent on the action of the wild-type LF4 gene product as demonstrated by the observation that triple mutants of lf4 lf1 and lf2 have long flagella (ASLESON and LEFEBVRE 1998).
The mechanisms that regulate flagellar length are being identified by cloning and characterizing each of the genes that can be mutated to produce a long-flagella phenotype. The LF4 gene product is a MAP kinase with high sequence similarity to a mammalian MAP kinase of unknown function called MOK (BERMAN et al. 2003). The LF3 gene product is a large (133 kD) protein with no sequence homologs in other organisms. LF3p localizes to small foci scattered throughout the cytoplasm, with little or no LF3p localizing to the flagella (TAM et al. 2003). LF2 encodes a cytoplasmically localized serine-threonine protein kinase (L. W. TAM, N. W. WILSON and P. A. LEFEBVRE, unpublished observations).
In this report we describe the cloning and characterization of the first length control gene to be identified genetically, LF1. The only LF1 mutant allele, lf1, was identified in a motility screen following chemical mutagenesis (MCVITTIE 1972a). We used a positional cloning approach employing an indexed bacterial artificial chromosome (BAC) library to clone the LF1 gene. LF1 encodes a novel protein found predominately in the cytoplasm in discrete foci similar to those containing LF3p. A very small amount of LF1p (<1%) localizes to the flagella.
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MATERIAL AND METHODS |
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Flagellar length measurements:
Cells fixed with 0.5% glutaraldehyde were observed using differential interference contrast (DIC) microscopy. Images were captured with a video camera and Scion Image 1.59 software, and flagellar lengths were measured using PhotoShop 5.5 and Scion Image 1.59 software. For flagellar regeneration experiments, cells were deflagellated using homogenization with a hand-held blender for 3 min. Deflagellation was confirmed by phase contrast microscopy. Cell samples taken before deflagellation, immediately following deflagellation, and at time points after deflagellation (30, 60, 90, 120, and 180 min) were fixed in 0.5% glutaraldehyde, and the lengths of 100 random flagella were measured per time point.
Cell swimming velocity measurements:
Swimming velocities from wild-type (137c) and lf1 mutant cells were measured as described by SAKAKIBARA et al. (1993). Average swimming velocity measurements were obtained for 25–30 samples per strain.
Chlamydomonas transformation:
An lf1 arg7 (A-A6) double-mutant strain was cotransformed with plasmid pARG7.8 DNA containing the complete argininosuccinate lyase gene (DEBUCHY et al. 1989) and BAC or plasmid DNA using the glass bead method (KINDLE 1990). Cells (5 x 107) were treated with Chlamydomonas autolysin for 45 min at room temperature under bright light. Cells were lightly pelleted and resuspended in 0.3 ml TAP media. Two to five micrograms of pARG7.8 DNA, 5–10 µg of BAC DNA (or 2–5 µg plasmid DNA), 5% PEG 8000, and 0.3 ml acid-treated glass beads were added to cells and vortexed together at top speed for 45 sec. TAP media (1 ml) was added to cells, and the entire volume of cells was plated on 1.2% TAP agar plates and placed in bright, constant light for 1 week. Transformed Arg+ colonies were picked individually into liquid M media and screened for rescue of the long-flagella phenotype using a Zeiss stereomicroscope to screen for normal swimming motility. The cotransformation efficiency rate was 
1–5% using BAC clones.
Cloning and sequencing of LF1:
DNA probes Dhc4 and Dhc5 (kindly provided by Mary Porter, University of Minnesota, Minneapolis), GSP1, and GP225 were labeled with [32P]dCTP by random primer labeling to use as hybridization probes for screening a BAC library of Chlamydomonas genomic DNA (available from the Clemson University Genomics Institute, Clemson, SC). Comparing restriction patterns of BAC clones digested with HindIII and BamHI, we identified BAC end fragments; fragments that were not common between BAC clones were assumed to be at the end of the BAC clone. BAC end fragments were purified from 0.8% TAE agarose gels using the GeneCleanII kit (Bio101 Systems, Vista, CA) and prepared for hybridization by random primer labeling. The smallest fragment found to be unique to a BAC clone was used to screen the library. Hybridization of the BAC library filters was performed at 42°, overnight, in 50% formamide, 5x SSPE, 10x Denhardt's, 1% SDS, and 300 µg/ml salmon sperm DNA followed by one wash at 42° in 2x SSPE, 1% SDS buffer, and three washes at 65° in 0.2x SSPE, 0.2% SDS buffer (SAMBROOK et al. 1989). DNA from BAC clones hybridizing to the labeled probes was isolated by alkaline lysis. BAC DNA was transformed into the A-A6 lf1 arg7 strain using the glass bead method described above, and rescue of the lf1 phenotype was determined by visual screens for normal swimming motility.
A 7.2-kb subclone (p7.2BB) containing the entire LF1 gene was obtained by digesting BAC 13m9 with BamHI and ligating the resulting fragment into pBluescript KS+. The subclone was sequenced on both DNA strands (Advanced Genetics Analysis Center, University of Minnesota, St. Paul) using gene-specific primers, and the DNA sequence was assembled using the Genetics Computer Group software (Madison, WI). Subsequent subclones were cloned into pBluescript KS+ (Figure 4) and tested for their ability to rescue the lf1 phenotype upon transformation. The sequence of the lf1 mutant allele was obtained by sequencing overlapping PCR amplification products that covered the entire LF1 coding region using gene-specific primers and the Epicentre (Madison, WI) Failsafe PCR kit, following the manufacturer's protocol. Eight independent PCR reactions from three independent DNA preparations confirmed the single-base-pair mutation in DNA from the lf1 mutant.
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RNA analysis:
Total RNA was isolated using the LiCl precipitation method of WILKERSON et al. (1994) except that 100 µg/ml of proteinase K was used in the lysis buffer. To obtain RNA from deflagellated cells, wild-type cells were deflagellated by pH shock (WITMAN et al. 1972) and allowed to regrow their flagella for 15, 30, 45, or 60 min before RNA isolation. Poly(A) RNA was isolated using Magnetight oligo(dT) particles (Novagen, Madison, WI) starting with 1 mg of total RNA. Poly(A) RNA (4–5 µg/lane) was size fractionated in a 1% MOPS-formaldehyde agarose gel (SAMBROOK et al. 1989) and transferred to Brightstar Plus membrane (Ambion, Austin, TX). cDNA probes were labeled by random priming with [32P]dCTP, and the membrane was hybridized in ULTRAHyb solution (Ambion) overnight at 42°, washed in 2x SSC and 0.2x SSC solutions containing 0.2% SDS at 65°, and exposed to X-ray film. Hybridization with two cDNA fragment probes, one from the 5' end and the other from the 3' end, confirmed that a single transcript was detected for LF1. Membranes were rehybridized using labeled DNA from the CRY1 gene [encoding the ribosomal protein S14 (NELSON et al. 1994)] as a loading control.
HA epitope tagging, immunoblot, and immunofluorescence analysis:
A triple HA tag [described by SILFLOW et al. (2001)] was inserted at the second SmaI site of the SmaI-BamHI(p6.6SB) subclone. Rescue of the lf1 phenotype by transformation with a plasmid having the HA tag in the forward orientation was observed. A plasmid with the HA tag in the reverse orientation was unable to rescue the lf1 phenotype, presumably due to the introduction of premature stop codons. To observe expression of the HA-tagged LF1 protein (HA-LF1p), immunoblot analysis was performed using the method of WILSON et al. (1999) with modifications. To analyze HA-LF1p expression in whole cells, 2 x 106 cells per sample were pelleted and resuspended in 1x SDS sample buffer (10% glycerol, 2% SDS, 0.1% bromphenol blue, 0.1 M dithiothreitol, and 0.0625 M Tris, pH 6.8), boiled for 5 min, and separated on an 8% acrylamide gel (20 mA, 1.5 hr). Protein was transferred to Immobilon P membranes (Millipore, Bedford, MA) in a buffer containing 192 mM glycine, 20% methyl alcohol, and 25 mM Tris, pH 8.3, at 102 V for 1 hr at room temperature using the Mini Transblot electrophoresis transfer cell (Bio-Rad, Hercules, CA). The membrane was fixed in 0.2% glutaraldehyde in 1x TBS (137 mM NaCl, 20 mM Tris, pH 7.6) for 45 min at room temperature, washed two times in 1x TBS, and blocked overnight at 4° in TBST (137 mM NaCl, 20 mM Tris, pH 7.6, and 0.05% Tween 20) containing 5% Carnation dry milk. After blocking, the membrane was rinsed twice with TBST and incubated with anti-HA antibody 3F10 (Roche, Indianapolis) at 1:1000 dilution in TBST containing 3% dry milk for 1 hr at room temperature. After the primary antibody incubation, the membrane was washed three times for 5 min each in TBST, incubated with anti-rat POD (Roche) antibody at 1:1000 dilution in TBST containing 3% dry milk for 1 hr at room temperature, and washed three times for 7 min each in TBST. The protein was detected using the ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ). To analyze HA-LF1 protein expression in both cell bodies and flagella, cells were deflagellated by pH shock and flagella were isolated as described by WITMAN (1986) except flagella and cell bodies were resuspended in 10 mM HEPES, pH 7.0. As a control for contamination of isolated flagella by cell body proteins, the membrane was incubated with an antibody to OEE1, a chloroplast protein, (kindly provided by Bridgette Barry, University of Minnesota, St. Paul; MAYFIELD et al. 1987) at a 1:400 dilution.
Immunofluorescence was performed using the method of SANDERS and SALISBURY (1995) with cells fixed in 4% paraformaldehyde, 10 mM HEPES, or methanol (–20°). We used anti-HA antibodies (Roche) at a 1:200 dilution and anti-rat antibodies conjugated to the Alexa 488 fluorochrome (Molecular Probes, Eugene, OR) at a 1:200 dilution or anti-
tubulin (kindly provided by Carolyn Silflow, University of Minnesota, St. Paul) at a 1:1000 dilution and anti-rabbit antibodies conjugated to Texas red (ICN, Aurora, OH) at a 1:200 dilution. Cells were mounted as described in the Slowfade Light Antifade kit (Molecular Probes) and observed with a Nikon800 microscope. Digital images were collected using a CoolCam liquid-cooled, three-chip color CCD camera and captured using Image Pro Plus v4.0 software.
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RESULTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LF1 cloning and characterization:
Although we have generated many different motility mutants using insertional mutagenesis, including >10 insertional alleles of lf4 (TAM and LEFEBVRE 1993; SMITH and LEFEBVRE 1996; ASLESON and LEFEBVRE 1998), no insertional alleles of lf1 have been isolated. We therefore obtained the LF1 gene by performing a chromosome walk using molecular markers near LF1 as probes to screen an indexed BAC library of Chlamydomonas DNA. The lf1 mutation maps to the right arm of linkage group II between ac12 and pf12 (MCVITTIE 1972b). Beginning with dynein heavy chain clones Dhc4 and Dhc5 (PORTER et al. 1996) as hybridization probes, we performed a chromosome walk that spanned an 740-kb region of linkage group II (Figure 2). Using molecular markers GSP1, GP225, and GP336 (KATHIR et al. 2003) and numerous BAC end fragments as probes, the BAC library was screened 20 times. A total of 101 BAC clones were identified and assembled into a contig of >740 kb of genome sequence. Seventeen clones spanned the entire length of the contig. Each of the 17 clones was introduced into lf1 cells by cotransformation with a selectable marker gene and transformed lines were screened for rescue of the lf1 phenotype. Both the flagellar length defect and the flagellar regeneration defect of lf1 cells were rescued by transformation with BAC clones 3b19 and 34f11. Mutant cells rescued by transformation had flagella of wild-type length (Figure 3A) and regenerated their flagella within 2 hr after deflagellation, as seen for wild-type cells (Figure 3B).
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3.5 map units (MCVITTIE 1972b). The corresponding physical distance is 300 kb, indicating that in this genomic region one genetic map unit (1 cM) corresponds to 85 kb.
A 7.2-kb BamHI genomic fragment (p7.2BB) was subcloned and shown to rescue the lf1 defect upon transformation. Examination of the DNA sequence of this clone using the GenScan and GeneMark gene prediction programs revealed a single open reading frame (ORF) of 804 amino acids. cDNA library screening and RT-PCR confirmed the exon/intron boundaries (Figure 4). LF1 encodes a glycine-rich (21%), novel protein with a predicted molecular weight of 80 kD and a pI of 7.9 (Figure 5A). Searches of databases showed no homology to any known protein and no conserved functional motifs. The lf1 allele was sequenced to reveal a single transversion mutation (G to T) resulting in an amber stop codon at amino acid 87 (Figure 5B). The resulting truncated protein would be 10% the length of the wild-type protein.
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Using probes from the 5' and the 3' ends of the LF1 gene, we measured transcript levels in wild-type, lf1, and lf1 rescued cells (Figure 5C). The 3.1-kb transcript was present in all strains, but the LF1 expression level was 60% lower in the lf1 mutant than in wild-type cells. This result would be expected for the lf1 amber mutation if Chlamydomonas uses the nonsense-mediated decay mechanism for degrading untranslatable mRNAs seen in many eukaryotic systems (reviewed by CULBERTSON and LEEDS 2003). The transcript was restored to wild-type levels in the lf1 rescued strain. Chlamydomonas cells upregulate the transcripts of many flagellar proteins after the cells are deflagellated. Using poly(A) RNA isolated from wild-type cells both before (lane P, Figure 5D) and at 15, 30, 45, and 60 min after deflagellation (Figure 5D), we found that the 3.1-kb LF1 transcript was not upregulated after flagellar amputation. A probe for the CRY1 gene encoding the ribosomal protein S14, whose transcript level is unaffected by deflagellation, was used as a loading control.
LF1p localization:
Attempts to generate antibodies to the LF1 protein have not been successful. As an alternative approach, we tagged the LF1 protein with a triple HA epitope for immunolocalization experiments (Figure 6A). Transformation of HA-LF1 gene constructs into lf1 cells rescued the lf1 phenotype. When the epitope tag was inserted in the reverse orientation, rescue of the lf1 phenotype was not obtained, presumably due to the introduction of multiple stop codons. To observe HA-LF1p expression, we performed an immunoblot of proteins from whole cells using four different transformed strains that had been rescued with the HA-tagged construct. A protein of 120 kD was observed in transformed strains that was not expressed in strains lacking the HA tag (Figure 6B). We compared levels of HA-LF1p in whole cells, cell bodies, and isolated flagella by loading equal amounts of protein (Figure 6C, lanes 1, 2, and 3). HA-LF1p was detected in each fraction. Incubation of the blot with an antibody to a chloroplast protein (OEE1p; MAYFIELD et al. 1987) that should not be present in the flagella showed that the isolated flagella were not contaminated by cytosolic proteins. To determine the relative amounts of HA-LF1p in the flagella vs. the cell bodies, dilutions of flagellar protein samples were examined. When protein samples from whole cells, cell bodies, and flagella from equivalent numbers of cells (2 x 106) were loaded on the gel, no band corresponding to HA-LF1p was detected in the lane containing isolated flagella (Figure 6C, lanes 4, 5, and 6). If a flagellar protein sample from 2 x 109 cells (Figure 6C, lane 10) was compared with a whole-cell or cell-body protein sample from 2 x 106 cells (Figure 6C, lanes 4 and 5), equivalent amounts of HA-LF1p were detected, indicating that 1000 times more LF1 protein is present in the cell body than in the flagella.
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DISCUSSION |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Potential interactions of LF1p with other LF gene products:
Several lines of evidence suggest that the LF1 gene product interacts with the products of the LF2 and LF3 genes in the regulation of flagellar assembly and length (MCVITTIE 1972a,b; BARSEL et al. 1988). Not only do double mutants of lf1 with lf2 or lf3 show a synthetic flagella-less phenotype, but also all three gene products are similarly localized in discrete spots in the cytoplasm with very little in the flagella (Figure 6D; TAM et al. 2003). LF1p and LF3p also cofractionate at 11s upon sucrose gradient centrifugation of cytoplasmic extracts (TAM et al. 2003). Triple mutants of lf4, lf1, and lf2-3 had long flagella, suggesting that LF4 may act downstream of LF1, possibly to induce flagellar shortening (ASLESON and LEFEBVRE 1998). LF4 also acts downstream of LF1 in the control of flagellar regeneration. lf1 lf4 double mutants regenerate flagella after amputation with the rapid kinetics of wild-type cells, whereas lf1 mutants alone regenerate flagella very slowly. This result suggests that the wild-type LF4 gene product is required to produce the flagellar regeneration defects in lf1 mutants. LF4 has recently been shown to encode a novel MAP kinase (BERMAN et al. 2003), suggesting the possible involvement of LF1 in the upstream portion of a signal transduction cascade involved in flagellar length control.
It is interesting that only 50% of the protein, from the amino terminus, is needed to rescue the lf1 phenotype upon transformation; the carboxyl end of the protein seems dispensable. The IC140 protein of Chlamydomonas axoneme acts in a similar manner (PERRONE et al. 1998) with the first 283 amino acids of the protein being dispensable for function. Perrone et al. concluded that the carboxyl terminus is sufficient to assemble a dynein protein complex. The carboxyl terminus of LF1p is not essential for function, but perhaps it helps to stabilize the protein. The protein is glycine rich (21%) especially within the carboxyl terminus. Glycine-rich regions are thought to be sites of protein:protein interactions that help with stabilization and flexibility of protein complexes (SACHETTO-MARTINS et al. 2000). The C terminus of LF1p may interact with other length control proteins, most likely LF2 and/or LF3, to form a cytoplasmic complex, visualized as discrete foci by immunofluorescence.
It seems likely that the single available mutant allele of lf1 is a null allele. The genetic lesion in lf1 generates a stop codon at amino acid 87, and there are no methionine codons for potential downstream reinitiation within the following 380 codons. If lf1 is a null allele, it is somewhat surprising that no new lf1 alleles were found among >10 long-flagella mutants identified in insertional mutagenesis screens (ASLESON and LEFEBVRE 1998). All insertional mutants with the long-flagella phenotype to date have proven to be lf4 alleles. Insertional null alleles of lf3 (TAM et al. 2003) and lf2 (L. W. TAM and P. A. LEFEBVRE, unpublished observations) have an "unequal-length flagella" or Ulf phenotype. Populations of Ulf mutants have many flagella-less cells; those cells with flagella often have flagella of unequal length, and a few cells have long flagella. This phenotype is clearly different from the long-flagella phenotype of the lf1 mutant, as well as putative hypomorphic alleles of lf2 and lf3. It is possible that LF4 is a hotspot for plasmid insertion during transformation or that LF1 is in an unfavorable genomic environment for plasmid insertion.
Control of flagellar length:
No detailed molecular models have yet been advanced to explain how Chlamydomonas cells can recognize the length of their flagella and maintain them at an equal and fixed length. Recently a proposal has been advanced that length regulation is a consequence of a balance between rates of assembly and disassembly of the doublet microtubules of the axoneme and that this balance is a consequence of a limiting supply of IFT proteins in the flagella (MARSHALL and ROSENBAUM 2001). Two different long-flagella mutants, lf3 (TAM et al. 2003) and lf1 (PERRONE et al. 2003), have now been shown to accumulate IFT proteins into their flagella above the levels seen in wild-type flagella. These proteins appear to accumulate in bulges at the tips and to a lesser extent along the length of the flagella. These bulges contain accumulations of IFT protein particles. Perhaps one role for the proteins in the cytoplasmic complex containing LF1p, LF2p, and LF3p is to control the partitioning of IFT protein components into and out of the flagella.
Positional cloning using BAC clones is feasible in Chlamydomonas: We determined, by obtaining rescue with BAC clones of both the pf12 and the lf1 mutations, that one map unit corresponds to 85 kb of genomic DNA. DUTCHER et al. (2002) performed an 720-kb BAC walk in their cloning of the Chlamydomonas bld2 gene, which encodes epsilon tubulin, and determined that in the relevant portion of linkage group III one map unit corresponds to 160 kb.
Our results highlight the ease with which BAC clones can be used for positional cloning of mapped genes. The transformation efficiency with BAC clones in Chlamydomonas cells is 1–5%, and transformation and phenotypic screening of transformed strains can be completed in 7–10 days. A molecular map containing >280 markers with an average spacing of 3–4 MU has been aligned with the genetic map (KATHIR et al. 2003). BAC contigs have been constructed around many of these markers. By using a nearby molecular marker as a starting point, positional cloning of genes identified by mapped mutations should become routine using the BAC library. The Chlamydomonas genome has been sequenced, and the draft sequence is available at the Chlamydomonas web site of the Joint Genome Institute (JGI) of the Department of Energy (http://genome.jgi-psf.org/chlre2/chlre2.home.html). The sequence of the ends of all 15,000 BAC clones in the indexed library is also available at the JGI site. With the availability of the genomic sequence and BAC clones, as well as the ease and efficiency of cotransformation, positional cloning of any gene on the basis of its position on the genetic map becomes possible.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIAL AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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