Erlangen-Nürnberg, Univ., Diss., 2009.

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.

Proefschrift Universiteit Maastricht. / Met lit. opg. - Met samenvatting in het Nederlands.

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.