The nucleotide sequences of the initiator transfer RNAs from bean cytoplasm and chloroplasts

The initiator tRNAsMet from the cytoplasm and chloroplasts of Phaseolus vulgaris have been purified and sequenced. The sequence of bean cytoplasmic initiator tRNAiMet is : pA-U-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-C-G-G-A-A-G-C-G-U-m2G-G-U-G-G-G2-C-C-C-A-U-t6A-A-C-C-C-A-C-A-G-m7G-D-m5C-C-C-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-Gm-G-C-U-C-U-G-A-U-A-C-C-AOH. The sequence of bean cytoplasmic tRNAiMet is almost identical to that of wheat germ and shows a high degree of homology with other cytoplasmic initiator tRNAs. The sequence of bean chloroplast initiator tRNAfMet is : pC-G-C-G-G-A-G-U-A-G-A-G-C-A-A-C-U-U-Gm-G-D-A-G-C-U-C-G-C-A-A-G-G-C-U-C-A-U-A-A-C-C-U-U-G-A-A-m7G-acp3U-U-A-C-G-G-G-T-psi-C-A-A-A-U-C-C-C-G-U-C-U-C-C-G-C-A-A- C-C-AOH. Bean chloroplast initiator tRNAfMet sequence shows procaryotic characteristics at the 5' end of the acceptor stem and in the TpsiC loop, but also contains some distinctive features.     

Recognition of the initiator tRNA by the Pseudomonas aeruginosa methionyl-tRNA formyltransferase: importance of the base-base mismatch at the end of the acceptor stem

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) in eubacteria is catalyzed by methionyl-tRNA formyltransferase (MTF). Features of the Escherichia coli tRNAfMet that are important for formylation are the base-base mismatch between nucleotides 1 and 72, and the second and third base pairs of the acceptor stem. The base-base mismatch is the most crucial formylation determinant in the E. coli tRNAfMet. However, it is not known whether this feature is also important for formylation of other eubacterial tRNAfMet. We cloned the Pseudomonas aeruginosa MTF gene by complementation of an E. coli MTF mutant strain with a genomic library, and investigated the catalytic properties and substrate specificity of the enzyme. The results show that the P. aeruginosa and E. coli enzymes have comparable affinities for the tRNAfMet and N10-formyltetrahydrofolate (fTHF) substrates. Overproduction of the P. aeruginosa MTF rescued the initiator activity of an E. coli formylation-defective tRNAfMet with a base pair between nucleotides 1 and 72, indicating that the base-base mismatch is utilized by the P. aeruginosa MTF for recognition of the tRNAfMet. Therefore, this feature may be used by MTFs from other eubacteria to distinguish the initiator from elongator tRNAs.     

Nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8.

The nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAf1Met and tRNAf2Met. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAfMet, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAfMet: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAfMet is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAf2Met has a reversed G-C pair at positions 52 and 64 compared with those in tRNAf1Met and E. coli tRNAfMet. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.     

Locations and sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC): the tRNAGly contains only two base-pairs in the D stem.

The nucleotide sequences of tobacco chloroplast genes for tRNAPro(UGG), tRNATrp, tRNAfMet and tRNAGly(GCC) have been determined. None of these genes contains an intron. One unusual feature is that the tRNAGly contains only two base-pairs (A-U, G-U) in the D stem. These four tRNA genes were located in the known physical map of tobacco chloroplast DNA. Hybridization analysis to chloroplast tRNA revealed that all four tRNA genes are transcribed in vivo.     

Biochemical research on oogenesis. Nucleotide sequence of initiator tRNA from oocytes and from somatic cells of Xenopus laevis.

The nucleotide sequence of initiator tRNA, tRNAfMet, from vitellogenic oocytes of Xenopus laevis was determined. The sequence was deduced from analysis of all T1 and pancreatic oligonucleotides and comparison with the sequence of initiator tRNA from other animal species. At least 80% of all initiator tRNA molecules from oocytes have the same nucleotide sequence. This means that most and probably all initiator tRNA genes which are active in oocytes are identical to one another. No structural difference was observed between liver and oocyte initiator tRNAs. Initiator tRNA from X. laevis has the same nucleotide sequence as initiator tRNA from several species of mammals. The genes coding for this RNA have therefore remained unchanged in the mammalian and amphibian lines for at least 300000000 years.     

Localization of noncovalently bound ethidium in free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer

Ethidium binds tRNAfMet with 17-fold enhancement in the emission intensity at 600 nm. Fluorescence titration of tRNAfMet with ethidium indicates a single high-affinity site in tRNAfMet with a dissociation constant of 5 microM. Ethidium is apparently rigidly bound to tRNAfMet and effectively shielded from solvent. tRNAfMet(8-13), tRNAfMet(3'-Flc), and tRNAfMet(D-PF) with fluorophores at thiouridine, the 3'-terminus, and dihydrouridine, respectively, are prepared, and the singlet-singlet energy-transfer efficiencies between these fluorophores and noncovalently bound ethidium are determined. The transfer efficiency between bound ethidium and the fluorophore in tRNAfMet(8-13) determined by donor quenching and sensitized emission is the same, strongly suggesting that there is only one bound ethidium per tRNAfMet molecule. The apparent distances between ethidium and various fluorophores including 3'-fluorescein, the 8-13 photo-cross-link, and D-proflavin are 41, 19, and 30 A, respectively, assuming random orientation between the donor and the acceptor. The results suggest that noncovalently bound ethidium is intercalated in the amino acid acceptor stem. In the complex of tRNAfMet and methionyl-tRNA synthetase, the transfer efficiencies for the tRNAfMet(8-13), tRNAfMet(3'-Flc), and tRNAfMet(D-PF) are reduced, enhanced, and little changed, respectively. These methionyl-tRNA synthetase induced changes suggest changes in the conformation of the 3'-terminal unpaired bases and the relative orientation or location between tRNAfMet and ethidium upon binding of methionyl-tRNA synthetase.     

Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.     

tRNA recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that anticodon bases are essential for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase (MetRS) [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759] and that the enzyme tightly binds to C34 at the wobble position of E. coli initiator methionine tRNA (tRNAfMet) [Pelka, H., & Schulman, L. H. (1986) Biochemistry 25, 4450-4456]. We have also previously demonstrated that an affinity labeling derivative of tRNAfMet can be quantitatively cross-linked to the tRNA binding site of MetRS [Valenzuela, D., & Schulman, L. H. (1986) Biochemistry 25, 4555-4561]. Here, we have determined the site in MetRS which is cross-linked to the anticodon of tRNAfMet, as well as the location of four additional cross-links. Only a single peptide, containing Lys465, is covalently coupled to C34, indicating that the recognition site for the anticodon is close to this sequence in the three-dimensional structure of MetRS. The D loop at one corner of the tRNA molecule is cross-linked to three peptides, containing Lys402, Lys439, and Lys596. The 5' terminus of the tRNA is cross-linked to Lys640, near the carboxy terminus of the enzyme. Since the 3' end of tRNAfMet is positioned close to the active site in the N-terminal domain [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180], this result indicates that the carboxy ends of the two polypeptide chains of native dimeric MetRS are folded back toward the N-terminal domain of each subunit.     

Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase

A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNAfMet). Incubation of the affinity-labeling tRNAfMet derivative with E. coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme. A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated. Cross-linking was effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAPhe. The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography. The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography. Two major peptide products were isolated plus several minor peptides. N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS. Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.     

Isolation and characterization of two 5-fluorouracil-substituted Escherichia coli initiator methionine transfer ribonucleic acids

Escherichia coli initiator methionine tRNA labeled in vivo with 5-fluorouracil (FUra) has been isolated and characterized. The tRNA, with essentially all its uracil and uracil-derived minor bases replaced by FUra, was purified by sequential chromatography, first on diethylaminoethylcellulose (DEAE-cellulose), at pH 8.9, followed by chromatography on Sepharose 4B, using a reverse salt gradient, then on DEAE-Sephadex A-50, and finally on benzoylated DEAE-cellulose. The last step resolved two FUra-substituted tRNAfMet-iso-accepting species, each with a specific activity over 1500 pmol/A260. Kinetic analysis shows both are aminoacylated at the same rate; apparent KmS for the two are 0.92 and 0.94 microM, compared with 1.7 microM for normal tRNAfMet. Chromatographic differences between the two forms of fluorinated tRNAfMet persist after aminoacylation, and the two tRNAs are not interconverted by denaturation and renaturation. The isoacceptors have nearly identical nucleoside composition, and both contain 7-methylguanosine and 2'-O-methylcytidine as the only modified nucleosides. Analysis of complete RNase T1 digests of the two methionine tRNAs shows that they differ in only one oligonucleotide. The sequence 20FpApGp, derived from the dihydrouridine loop and stem region, which is found in one of the isoaccepting forms of the tRNA, is replaced by an oligonucleotide containing adenine and guanine, but no FUra in the other. A modified FUra, with the properties of a 5-fluoro-5,6-dihydrouracil derivative, is detected in this tRNA. 19F NMR spectra of the two species of FUra-substituted initiator tRNA show 9-10 resolved resonances for the 12 FUra residues incorporated. The spectra differ primarily in the shift of one peak in the form lacking the sequence 20FpApGp, from 4.8 ppm downfield from free FUra (= 0 ppm) to 14.9 ppm upfield from the standard.     

The Pseudomonas aeruginosa initiation factor IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa

Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination.     

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase

Derivatives of E. coli tRNAfMet containing single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with RNase A. RNA ligase is then used to join each of four trinucleotides, NAU, to the 5' half molecule and to subsequently link the 3' and modified 5' fragments to regenerate the anticodon loop. Synthesis of intact tRNAfMet containing the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMet with respect to its ability to be aminoacylated by E. coli methionyl-tRNA synthetase. Substitution of any other nucleotide at the wobble position of tRNAfMet drastically impairs the ability of the synthetase to recognize the tRNA. Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMet is four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base. In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme. These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminoacylation of tRNAfMet.     

Topographic modeling of free and methionyl-tRNA synthetase bound tRNAfMet by singlet-singlet energy transfer: bending of the 3'-terminal arm in tRNAfMet

Conformations of tRNAfMet, free and methionyl-tRNA synthetase bound forms, are analyzed by using singlet-singlet energy transfer as a spectroscopic ruler. tRNAfMet(8-13,3'-Flc), tRNAfMet(8-13,D-Etd), and tRNAfMet(3'-Flc,D-Etd) are prepared by sequential chemical modifications. The methionyl-tRNA synthetase binding affinity of these double-labeled tRNAfMets is similar to those of unmodified tRNAfMet. The fluorescence properties of the individual fluorophore in these tRNAs, including emission spectra, anisotropy, and quenching by methionyl-tRNA synthetase, are similar to those of single-labeled tRNAfMet. The transfer efficiencies of double-labeled tRNAfMets, as determined by both donor quenching and sensitized emission, showed efficient energy transfer in all cases. Random orientation being assumed, the apparent distances are 25 A between 8-13 and D20, 44 A between 8-13 and the 3'-terminus, and 49 A between the 3'-terminus and D20, respectively, in free tRNAfMet. Upon binding of methionyl-tRNA synthetase, the apparent distances are 25 A between 8-13 and D20, 45 A between 8-13 and the 3'-terminus, and 54 A between the 3'-terminus and D20, respectively. These results provide topographic models of these specific locations in free and methionyl-tRNA synthetase bound tRNAfMet and suggest that the immobilized 3'-terminal arm in the amino acid acceptor stem bends toward the inner loop of the L-shaped tRNA upon binding of methionyl-tRNA synthetase.     

Structural features that underlie the use of bacterial Met-tRNAfMet primarily as an elongator in eukaryotic protein synthesis

Met-tRNAfMet from Escherichia coli is utilized efficiently as an elongator tRNA during protein synthesis in the rabbit reticulocyte lysate since it rapidly incorporates its methionyl residue into the same tryptic peptides of rabbit globin as the endogenous Met-tRNAmMet. Therefore, it must lack the structural characteristics that prevent the eukaryotic initiator tRNA from entering elongation. In contrast, E. coli Met-tRNAfMet appears to initiate very poorly since, unlike reticulocyte Met-tRNAiMet, it forms no detectable 43 S preinitiation complexes, and only a very small fraction of the methionine it contributes to polyribosomal peptidyl-tRNA is found at the N terminus. The bacterial fMet-tRNAfMet, which cannot elongate, is utilized for polypeptide chain initiation at a much lower level than the formylated Met-tRNAiMet from eukaryotes. The ability of E. coli Met-tRNAfMet to be used as an elongator and fMet-tRNAfMet as an initiator in the reticulocyte lysate may be considerably underestimated because of the rapid enzymatic hydrolysis of these initiator tRNAs in the lysate. The enzyme hydrolyzes fMet-tRNAfMet and Met-tRNAfMet from E. coli in a strictly Mg2+-dependent manner but not the corresponding species from yeast or rabbit reticulocytes. It also hydrolyzes yeast N-acetyl-Phe-tRNAPhe and reticulocyte peptidyl-tRNA, showing that this enzyme--like the eukaryotic protein synthetic machinery--does not readily distinguish the bacterial tRNAfMet from eukaryotic elongator tRNA.     

Interaction between Green and Far-Red Light on the Low Fluence Rate Chloroplast Orientation in Mougeotia

Continuous irradiation of Mougeotia with linearly polarized green light (550 nanometers, 0.2 watt per square meter) induces a change in the orientation of its chloroplast from profile to face position, if the electrical vector of the green light is vibrating normal to the cell axis. This change is complete within 25 minutes of the onset of irradiation. In contrast, if the electrical vector of the green light is parallel to the cell axis, no chloroplast reorientation is induced, even with a fluence rate as high as 3 watts per square meter. Furthermore, unpolarized far-red light (727 nanometers, 2 watts per square meter) given alone has no effect on chloroplast reorientation. Simultaneous and continuous irradiation with polarized green light, regardless of its plane of polarization, together with unpolarized far-red light, however, does lead to chloroplast reorientation. These data indicate that, in addition to the red-absorbing form of phytochrome, there exists in Mougeotia another sensory pigment absorbing green light.     

The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo. A mechanism for the inhibition of neural protein synthesis by phenylalanine.

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

Synthesis of 5' fragments of formylmethionine transfer ribonucleic acid and their reconstitution with a natural three-quarter molecule

An eicosanucleotide C--G--C--G--G--G--G--U--G--G--A--G--C--A--G--C--C--U--G--Gp corresponding to the bases 1--20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C--G--C--G, G--G--G--U--G--G and A--G--C--A--G--C--C--U--G--Gp using RNA ligase from T4-infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [gamma-32P]ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3'-three-quarter molecule obtained by limited digestion with RNase T1 of the natural tRNAfMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet.     

Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta)

The 22 published chloroplast genomes of green algae, representing sparse taxonomic sampling of diverse lineages that span over one billion years of evolution, each possess a unique gene arrangement. In contrast, many of the >190 published embryophyte (land plant) chloroplast genomes have relatively conserved architectures. To determine the phylogenetic depth at which chloroplast gene rearrangements occur in green algae, a 1.5-4 kb segment of the chloroplast genome was compared across nine species in three closely related genera of Trebouxiophyceae (Chlorophyta). In total, four distinct gene arrangements were obtained for the three genera Elliptochloris, Hemichloris, and Coccomyxa. In Elliptochloris, three distinct chloroplast gene arrangements were detected, one of which is shared with members of its sister genus Hemichloris. Both species of Coccomyxa examined share the fourth arrangement of this genome region, one characterized by very long spacers. Next, the order of genes found in this segment of the chloroplast genome was compared across green algae and land plants. As taxonomic ranks are not equivalent among different groups of organisms, the maximum molecular divergence among taxa sharing a common gene arrangement in this genome segment was compared. Well-supported clades possessing a single gene order had similar phylogenetic depth in green algae and embryophytes. When the dominant gene order of this chloroplast segment in embryophytes was assumed to be ancestral for land plants, the maximum molecular divergence was found to be over two times greater in embryophytes than in trebouxiophyte green algae. This study greatly expands information about chloroplast genome variation in green algae, is the first to demonstrate such variation among congeneric green algae, and further illustrates the fluidity of green algal chloroplast genome architecture in comparison to that of many embryophytes.