Bonn, Universiẗat, Diss., 1999.

D.Sc. / Please refer to full text to view abstract

In order to expand our understanding of the expression of chloroplast genes, the structure and expression of the Euglena gracilis rpoC1 and rpoC2 loci were studied. The rpoC1 and rpoC2 gene products are similar to the amino- and carboxyl-terminal regions of the $\beta\sp\prime$ subunit of E. coli RNA polymerase. The nucleotide sequence (7,270 bp) was determined for 100% of both strands encoding these two genes. The rpoC1 and rpoC2 genes are located downstream and in the same polarity as the rpoB gene. The organization of the Euglena rpoB-rpoC1-rpoC2 genes is conserved in plant chloroplasts and is similar to the E. coli rpoB-rpoC operon. The Euglena rpoC1 gene (586 codons) encodes a polypeptide with a predicted molecular weight of 68,043. The rpoC1 gene is interrupted by one group II intron of 349 bp, seven group III introns of 107, 100, 119, 97, 110, 102 and 103 bp, and three atypical introns of 210, 213 and 198 bp. The Euglena rpoC2 gene (830 codons) encodes a polypeptide with a predicted molecular weight of 94,628. The rpoC2 gene is interrupted by two group II introns of 580 and 514 bp, respectively. All of the exon-exon junctions were experimentally determined via cDNA cloning and sequencing analysis. Multiple protein alignments of the rpoC1 and rpoC2 gene products with related proteins from bacteria and chloroplasts were used to identify conserved regions. Transcripts from the rpoC1 and rpoC2 loci were characterized via Northern analysis. The rpoB, rpoC1 and rpoC2 genes are cotranscribed. Fully spliced tri-, di- and monocistronic transcripts were detected with hybridization probes specific for each gene. The relative abundance of the rpoC1 and rpoC2 transcripts is similar in RNA from dark- and light-grown Euglena. The mature 5'-ends of the rpoC1 and rpoC2 genes were mapped by primer extension. The 3'-end of the mature rpoC2 transcript was localized via an S1 nuclease protection assay. The rpoC1 and rpoC2 gene products were also compared to the largest subunits of RNA polymerases from archaebacteria and eukaryotes. The evolution of the Euglena genes is discussed.

The chloroplast genes of Euglena gracilis contain more than 100 introns. A comparison of intron content and position among plastid and prokaryote genes has led to the hypothesis that introns have been inserted into chloroplast genes during evolution. Several Euglena loci contain unusual introns. These introns have been characterized by direct primer extension cDNA sequencing, cDNA cloning and sequencing, and northern hybridization. The psbF locus has a 1042 nt intron that appears to be one group II intron inserted into domain V of another group II intron. It was determined that a 618 nt internal intron is first excised from the 1042 nt intron, resulting in a partially spliced pre-mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. The term "twintron" was used to define this new genetic element. Splicing of the internal and external introns occurs via lariat intermediates. The splicing of the 409 nt intron of the rps3 gene was also examined. This intron is a "mixed" twintron, composed of a 311 nt group II intron internal to a 98 nt group III intron. The splicing of four additional introns with mean lengths twice typical group III introns, three within the rpoC1 gene and one within the rpl16 gene, was analyzed. The 1604 nt intron in the psbC gene, which encodes orf458, was also examined. These introns are group III twintrons. Orf458 is encoded within the internal group III intron of the psbC twintron. Splicing of internal introns in three of the five group III twintrons involves multiple 5'- and/or 3'-splice sites. Excised group III introns accumulate as lariat RNAs. Twintrons represent evidence for intron insertion during gene evolution. One possible mechanism for twintron formation is by intron transposition. The disruption of functional domains by internal introns may necessitate a sequential in vivo splicing pathway, requiring excision of internal introns prior to excision of external introns. The origins of alternative splicing and a possible evolutionary relationship between group II, group III and nuclear pre-mRNA introns are discussed.

No description available.

The rpoB gene coding for a β-like subunit (homologous to the E. coli DNA-dependent RNA polymerase β subunit) of the chloroplast DNA-dependent RNA polymerase was located on the chloroplast genome of Euglena gracilis distal to the rrnC ribosomal RNA operon. The complete nucleotide sequence of the gene was determined. The sequence includes 97 bp of the 5S rRNA gene, an intergenic spacer of 1264 bp, the rpoB gene of 4249 bp, 84 bp spacer and 67 bp of the rpoC1 gene. The rpoB gene is of the same polarity as the rRNA operons. The organization of the rpoB and rpoC genes resemble the E. coli rpoB-rpoC and higher plants chloroplast rpoB-rpoC1-rpoC2 operons. The Euglena rpoB gene (1082 codons) encodes a polypeptide with predicted molecular weight of 124,288. The rpoB gene is interrupted by seven Group III introns of 93, 95, 94, 99, 101, 110 and 99 bp, respectively, and a Group II intron of 309 bp. All other known chloroplast rpoB genes lack introns. All the exon-exon junctions were experimentally determined by cDNA cloning and sequencing or direct primer extension RNA sequencing. Transcripts from the rpoB locus were characterized by Northern hybridization. Fully-spliced, monocistronic rpoB mRNAs, as well as rpoB-rpoC1 and rpoB-rpoC1-rpoC2 mRNAs were identified. Unspliced intron-containing transcripts could not be detected in these experiments. The rpoB gene is the first gene in the RNA polymerase rpoB-rpoC1-rpoC2 transcription unit. The three genes are transcribed from a promoter located upstream the rpoB gene. The transcript is processed to mature monocistronic mRNAs. The relative abundance of the mono-, di- and tricistronic mRNAs appear to be similar in RNAs isolated from photoautotrophic, heterotrophic and dark grown cells. The mature 5'- and 3'-ends of the mature rpoB monocistronic transcripts were determined via S1 nuclease mapping and primer extension RNA sequencing. In addition, the sequence of the 23S rRNA from the rrnC operon and the intergenic spacer between the rrnA and rrnB operon were determined. Transcription initiation for the ribosomal RNA transcription unit was determined via Northern analysis and S1 nuclease mapping of chloroplast RNA that was in vitro 5'-end labeled. Two transcription initiation sites were mapped at positions +1 and -50 upstream the 16S rRNA gene. The 3'-ends of the rrnA/rrnB and rrnC 5S rRNA were determined using S1 nuclease protection experiments. The protected fragments were of identical size. The rpoB-C1-C2 DNA sequence has been submitted to EMBL, accession number X17171, and the 23S rRNA DNA sequence was given the number X13310.

1. Distribution of the two photosystems (PS I and PS II) in the thylakoid membranes of the alga Euglena gracilis. The distribution of photosystem I and II (PS I and PS II) in the alga Euglena gracilis Z strain was studied by electron microscopic immunocytochemistry. In this alga, the thylakoids are not organized in gram structures, as they are in higher plants. Two different antibodies were used to identify PS I. One is directed against particles of PS I from maize and the other against the 60 and 62 kDa PS I reaction centre proteins of the cyanobacterium Synechococcus elongatus. Both antibodies demonstrated the presence of PS I in the two types of thylakoid membranes, appressed (AM) and non-appressed (NAM). Quantitative analysis showed that 60--74% of PS I is in the AM and 26--40% is in the NAM, and since about 80--90% of the membranes are AM, that PS I is more concentrated in the NAM. An antibody directed against the CP47 protein of PS II also revealed labelling in both types of thylakoid membranes (54% in AM and 46% in NAM). PS II is again more concentrated in the NAM. I demonstrated by the photo-oxidation of 3,3'-diaminobenzidine that there is PS I activity in the two types of membranes and, moreover, that there are changes in this activity during the light cycle of the cell. My results indicate that the distribution of PS I and PS II in Euglena gracilis Z strain is different from that of higher plants and is similar to that seen in green algae. The possible evolutionary significance of our observations are discussed. / 2. Localization of the protein CP47 (plastid protein) in the mitochondria of euglenoids. The localization of the CP47 protein to the mitochondria of euglenoids was studied by electron microscopic immunocytochemistry. My results demonstrate that this protein, which is coded by chloroplast DNA in all algae and plants, is present in whole or in part in the mitochondria of Euglena gracilis and related euglenoids. I used two different antibodies against the protein CP47 (anti-CP47 from Chlamydomonas reinhardtii and S. elongatus) to test wild-type, light-grown, cells of Euglena. Both antibodies selectively labelled the mitochondria. These results furthermore suggest that this labelling is particularly associated with mitochondrial cristae. Anti-CP47 from S. elongatus also labelled the mitochondria of other euglenoids, such as dark-grown cells of Euglena gracilis, the mutant Y9Z1NaL, and Astasia longa. Since the CP47 protein is present in dark-grown cells and in the mutant Y9Z1NaL, which are organisms that do not have an active psbB gene, I suggest that a gene transfer has occurred from the plastid to the mitochondria during evolution. Because our results show the presence of CP47 in the mitochondria of Astasia longa, I postulate that the transfer occurred before the branching of Astasia from Euglena.

M.Sc. (Zoology) / Axenic cultures of Euglena gracilis in different basal medium concentrations, to simulate eutrophic conditions, were exposed to different concentrations of Monocrotophos and Dichlorvos to determine the effect of the pesticides on the growth reaction. The effect of the pesticides on the chlorophyll a content of test organisms exposed to equivalent toxicities of pesticides was determined. The amount of pesticide accumulated and me tabolised was determined by the choline esterase method and by gaschromatography. More pesticide was found to be accumulated and metabolised by organisms in the lower concentration of basal medium.

The structure and expression of a novel Euglena gracilis chloroplast ribosomal protein operon was studied by gene mapping, molecular cloning, nucleotide sequencing primer extension and Northern analyses. The nucleotide sequence (12,240 bp) was determined for 100% of both strands encoding the 12 genes, rpl23 - rpl2 - rps19 - rpl22 - rps3-(2.8 kb region)- rpl16 - rpl14 - rpl5 - rps8 - rpl36 - trnI - rps14. The gene organization resembles the S10 and spc ribosomal protein operons of E. coli. The rpl5 gene was a new chloroplast gene not previously reported for any chloroplast genome nor described as a nuclear gene. The presence of numerous introns and an unusual 2.8 kb rps3-rpl16 intercistronic region were additional features that were unparalleled in other chloroplast DNAs. At least 15 introns were identified in the genes. Evidence is presented from primer extension analysis of chloroplast RNA for the correct in vivo splicing of six of the introns. Two introns within rps8 flanked an 8 bp exon, the smallest exon yet characterized in a chloroplast genome. Four introns shared structural properties with group II organelle introns. The remaining 11 introns were defined as new category of organelle intron, now designated "group III." The presence of additional introns in several intercistronic regions is proposed. Conserved regions in the predicted polypeptides were identified from the alignments with related proteins from other chloroplasts and bacteria. Evidence from Northern hybridization experiments with gene-specific probes supported the interpretation that 11 ribosomal protein genes, the 2.8 kb rps3-rpl16 intercistronic region and trnI were co-transcribed and encoded in a single operon. The co-transcription of genes coding for proteins and a tRNA is a novel finding for a chloroplast operon. Several stable polycistronic transcripts were identified, including a common 8.3 kb pre-mRNA. Stepwise processing pathways proposed for the mRNAs are described. Most mRNAs appeared to be fully spliced. The 5$\sp\prime$ ends of mRNAs for the first gene in the operon, rpl23, were mapped by primer extension. Plastid mRNAs from dark and light grown Euglena were analyzed on Northern blots.

Die Photoaktivierte Adenylatzyklase PAC ist in E. gracilis an der Phototaxis beteiligt und besteht aus den zwei unterschiedlich großen Proteinen PACalpha und PACbeta. Beide besitzen jeweils zwei FAD bindende (BLUF) Domänen F1 und F2 sowie zwei Zyklasedomänen C1 und C2. An den Zyklasedomänen findet die Umsetzung von ATP in cAMP statt und die BLUF-Domänen werden für die Lichtaktivierung benötigt. Für diese Arbeit wurde PAC und Mutanten davon heterolog in Oocyten von Xenopus laevis exprimiert. PAC besitzt bereits im Dunkeln Adenylatzyklaseaktivität, die durch Belichtung erhöht werden kann. Die Zunahme der Aktivität erfolgt mit einer Zeitkonstante von unter 100 ms, die Abnahme nach der Belichtung hat eine Zeitkonstante im Bereich von 10ms. Das für die katalytische Umsetzung in allen Klasse III Nukleotidzyklasen benötigte Dimer zweier Zyklasedomänen ist in PAC das Dimer aus C1 und C2. Durch Messungen mit PAC-Mutanten, bei denen jeweils eine Zyklasedomäne defekt war, konnte gezeigt werden, dass diese Dimerisierung in PACalpha intermolekular auftritt. Ebenso wurde gezeigt, dass ein solches Dimer aus Zyklasedomänen von PACalpha und PACbeta bestehen kann. Der Austausch der Zyklasedomänen von PACalpha durch Zyklasedomänen der Guanylatzyklasen GCY35 und GCY36 aus C. elegans führte zu einem Verlust der Zyklaseaktivität. Die Proteine wurden aber zumindest teilweise korrekt gefaltet, was durch Dimerbildung mit Knockoutmutanten von PAC in Koexpressionsexperimenten gezeigt werden konnte. Die Fusionsproteine aus PACalpha und den CNG-Kanälen CNGA2 und OLF führten in Oocyten zu einer deutlich geringeren Leitwertänderung als eine Expression der Einzelproteine. Sowohl bei einer N-terminalen Fusion des Kanals an PAC als auch bei der C-terminalen Fusion war es jeweils der Kanal, der im Fusionsprotein stark gehemmt war. Eine Deletion des C-Terminus von PACalpha führte zu einem nicht funktionsfähigen Protein, das auch in Koexpression mit PAC-Knockoutmutanten keine messbare Adenylatzyklaseaktivität zeigte. Wurde die F2-Domäne deletiert, so verlor PAC ebenfalls seine Zyklaseaktivität vollständig. Die C1-Domäne war aber korrekt gefaltet, was durch eine Koexpression mit PAC-Mutanten gezeigt werden konnte, die in einer ihrer Zyklasedomänen defekt waren. Beide Chimären aus PACalpha und PACbeta besaßen Adenylatzyklaseaktivität. Diese war bei der Chimäre mit dem C-terminalen Teil von PACalpha deutlich höher als bei der Chimäre mit dem C-terminalen Teil von PACbeta, was darauf hindeutet, dass im C-terminalen Teil von PAC der Grund für den Aktivitätsunterschied zwischen PACalpha und PACbeta liegt. Für die Veränderung der Substratspezifität von einer Adenylat- zu einer Guanylatzyklase waren Mutationen an mindestens drei Aminosäuren erforderlich. Die ebenfalls hergestellten Einzel- und Doppelmutanten verhielten sich wie der Wildtyp oder hatten eine deutlich eingeschränkte Adenylatzyklaseaktivität. Bei der Tripelmutante PACalpha K250E T319G S329Y war Guanylatzyklaseaktivität nachweisbar, die aber geringer war als die noch vorhandene Adenylatzyklaseaktivität. Die Quadrupelmutante PACalpha K250E D317K T319G S329Y zeigte ebenfalls lichtinduzierbare Adenylatzyklaseaktivität, die ca. 0,3% der Aktivität der Wildtyp-PACalpha entsprach. Die Guanylatzyklaseaktivität dieser Mutante war ca. dreifach höher als deren Adenylatzyklaseaktivität. Somit konnte gezeigt werden, dass sich durch die Mutation weniger einzelner Aminosäuren die Substratspezifität von PAC von ATP nach GTP verschieben lässt. / The photoactivated adenylyl cyclase PAC is involved in phototaxis in E. gracilis. It consists of two subunits of different size which are called PACalpha and PACbeta. Both of them harbour two FAD-binding domains (F1, F2) and two cyclase domains (C1, C2). PAC and mutants of PAC have been heterologously expressed in Oocytes of Xenopus laevis. Already in darkness PAC shows a basal level of adenylyl cyclase activity, which can be increased by illumination. The increase in cyclase activity occurs with a time constant lower than 100 ms, whereas the decrease after illumination has a time constant around 10 ms. The dimer of two cyclase domains, which is necessary for catalytic conversion in all class III cyclases, is formed of C1 and C2 in PAC. In electrophysiological experiments with PAC mutants which were defective in either of the cyclase domains it has been shown, that this dimer in PACalpha occurs intermolecularly. Furthermore it has been shown, that this dimer can occur between PACalpha and PACbeta. Mutants of PACalpha where the cyclase domains have been substituted by the cyclase domains of the guanylyl cyclases GCY35 and GCY36 from C. elegans lost their ability to produce cAMP. However coexpression experiments with PAC knockout mutants indicated correct translation of the substitution mutants. Expression of fusion proteins of PACalpha with the CNG channels CNGA2 and OLF showed less light-inducable conductance changes than the expression of the single protein. In both the N-terminal and C-terminal fusion of the channel to PACalpha it was the channel which was the most affected part of the fusion protein. Deletion of the C-terminus of PACalpha results in a non-functional protein, which in coexpression with PACalpha knockout mutants shows no measurable cyclase activity. When deleting the F2-domain, PACalpha also loses its cyclase activity completely. However, the C1-domain was transcribed correctly, which could be shown by coexpression with a C1-knockout mutant. Both PACalpha-PACbeta chimeras showed adenylyl cyclase activity. Whereas the activity in the chimera with the C-terminal part of PACbeta showed little cyclase activity, the chimera possessing the C-terminal part of PACalpha showed adenylyl cyclase acitvity, which was comparable to the wildtype of PACalpha. This indicates that the part of PAC which is responsible for the difference in cyclase activity between PACalpha and PACbeta must be present in the C-terminal half of PAC. For altering PAC’s substrate specificity it was necessary to mutate at least three amino acids. The single and double mutants of PACalpha which were generated resulted in wildtypelike behaviour or reduced adenylyl cyclase activity. The triple mutant PACalpha K250E T319G S329Y showed guanylyl cyclase activity which was lower than its remaining adenylyl cyclase activity. The quadruple mutant PACalpha K250E D317K T319G S329Y also showed adenylyl cyclase activity, which was about 0.3% of the activity in PACalpha wildtype. The guanylyl cyclase activity of this mutant was about threefold higher than its adenylyl cyclase activity. Thus, it could be shown, that by mutating few single amino acids the substrate specificity of PACalpha was shifted from ATP to GTP.