J.-D. Rochaix

Sequence of the chloroplast DNA region of Chlamydomonas reinhardii containing the gene of the large subunit of ribulose bisphosphate carboxylase and parts of its flanking genes

Abstract The transcriptional organization and the sequence of a 4000 base region of the chloroplast genome of Chlamydomonas reinhardii have been determined. The region includes the gene for the large subunit (LS) of ribulose bisphosphate carboxylase and portions of two neighbouring genes X and Y which are oriented in opposite direction relative to the LS gene. The transcripts of the LS, X and Y genes are 1600, 2200 and 2400 bases, respectively. The 5′ untranslated regions of the chloroplast messenger RNAs are variable in size: 91 ± 3 and 430 ± 10 bases for LS and X, respectively. The LS gene codes for a polypeptide of 475 amino acids whose sequence diverges 13 to 14% from the LS amino acid sequence of maize and spinach. The corresponding gene sequences differ 23 to 25% from each other. Most of the nucleotide differences occur in the third position of the codons and in the 3′ terminal portion of the gene. The three catalytic sites and the CO 2 activator region of the LS polypeptide have been highly conserved between C. reinhardii , spinach and maize. Only 40 different codons are used in the LS gene of C. reinhardii . The 5′ upstream regions of the LS and X genes contain sequences which resemble bacterial translational and transcriptional signals. Several repetitive elements are interspersed throughout the A + T-rich spacer between the X and LS genes. The 3′ untranslated region of the LS and Y mRNAs display stem-loop secondary structures which are reminiscent of bacterial terminators.

Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii

Abstract The chloroplast DNA of Chlamydomonas reinhardii has been examined by restriction endonuclease analysis. EcoRI, BamHI and BglII produce 30, 17 and 12 fragments, respectively, whose sites have been determined by electron microscopy and by comparative gel electrophoresis. These fragments have been ordered into a circular map which corresponds to a genome size of Mr = 126 × 106. The map was established by comparing the double digests of individual restriction fragments and by hybridizing purified labelled fragments to restriction enzyme digests of chloroplast DNA. The restriction fragments were isolated by molecular cloning or by preparative agarose gel electrophoresis. The two sets of chloroplast ribosomal RNA genes are contained within two inverted repeats of 13 × 106 molecular weight, which are located nearly at opposite sides of the map. In addition, the mapping studies have revealed the presence of short repeated base sequences which are interspersed throughout the chloroplast genome.

Anatomy of the chloroplast ribosomal DNA of chlamydomonas reinhardii

Abstract Two ribosomal DNA (rDNA) regions exist in the circular chloroplast genome of C. reinhardii. They are located at widely distant sites on the map in an inverted orientation. The chloroplast rDNA has been cloned and a fine structure map has been established. The small chloroplast ribosomal subunit contains 16S RNA, while the large subunit contains the four distinct RNA species 23, 7, 5 and 3S rRNA. The location of the rRNA genes has been determined by electron microscopy and by hybridization of the purified rRNAs to various rDNA restriction fragments. The gene order in the direction of transcription is 16, 7, 3, 23 and 5S. A spacer (Sp) of 1.68 ± 0.13 kb separates the 16S and the 7S RNA genes, and a very small spacer of 0.16 ± 0.04 kb separates the 7S and 23S RNA genes. The 5, 23, 3 and 7S RNAs are transcribed on the same strand. A surprising result is the interruption of all chloroplast 23S rRNA genes by a 940 base pair (bp) intervening sequence at a site 0.27 ± 0.04 kb distant from the 5′ end of the 23S RNA coding strand. This interruption has been observed both in cloned and uncloned chloroplast rDNA restriction fragments. When single-stranded chloroplast rDNA is mounted for electron microscopy, distinctive features of secondary structure which flank the rRNA gene sequences can be seen.

Ribosomal RNA gene amplification by rolling circles

Abstract Previous work has raised the possibility that gene amplification in Xenopus laevis oocytes involves a rolling circle intermediate (Hourcade et al., 1973a,b). We have combined electron microscopy with autoradiography in order to examine the structure of replicating ribosomal DNA molecules. The frequency of lariats (the presumptive rolling circles) in unlabelled ribosomal DNA is about 1%. If only labelled molecules are scored after a six-hour pulse with radioactively labelled DNA precursors, the proportion of lariats increases to about 8.5%. After pulses of two hours or less, the frequency rises still further to about 18%. In pulse and pulse-chase experiments, the lariats display a labelling pattern that is consistent with a rolling circle model: (1) as the pulse length is increased the labelled region in the tail grows from the replication fork towards the free end of the tail; (2) after a pulse-chase the labelled region is displaced towards the free end of the tail and no label remains associated with the lariat circle. The frequency of labelled free circles is lower by 80% in pulse-chased DNA than in DNA that has not been chased. This suggests that most of the radioactive circles are derived from broken rolling circles. Cyclization of lariat tails could account for the remaining labelled circles.

Characterization of the gene and messenger RNA of the large subunit of ribulose 1,5-diphosphate carboxylase in Chlamydomonas reinhardii

Abstract The gene coding for the large subunit of ribulose 1,5-diphosphate carboxylase (LS) of Chlamydomonas reinhardii has been cloned and localized at a unique site on the physical map of the chloroplast genome. It is contained within a 5.5 × 10 3 base Eco RI fragment which can act as template in vitro in a coupled transcription-translation system to produce a polypeptide, of the same size as LS, which is specifically immunoprecipitated with antibodies prepared against LS. The fingerprint of this in vitro synthesized polypeptide closely resembles that of cellular LS. The hybrid plasmid coding for LS inhibits specifically the in vitro translation of LS when it is hybridized to poly(A) − RNA prior to translation. The size of the LS messenger RNA has been estimated at 1.2 ± 0.3 × 10 3 bases by measuring R-loop structures in the electron microscope and two major species at 1.5 × 10 3 and 2.5 × 10 3 bases have been detected by hybridizing labelled LS-plasmid to cellular RNA fractionated on denaturing agarose gels.

Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii

Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus. Arginine prototrophy was used as a selective marker. The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E. coli after transformation. Four of them were characterized. Their inserts of 415, 257, 153, and 102 bp all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome. One of these plasmids also promotes autonomous replication in yeast. Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 bp that may play a role in promoting autonomous replication in C. reinhardii.

Localization and sequence analysis of chloroplast DNA sequences of Chlamydomonas reinhardii that promote autonomous replication in yeast

Four distinct chloroplast DNA segments from Chlamydomonas reinhardii of 400, 415, 730 and 2300 bp which promote autonomous replication in yeast have been mapped on the chloroplast genome. Plasmids carrying these chloroplast DNA fragments are unstable in yeast when the cells are grown under non‐selective conditions. Sequence analysis of three of these chloroplast ARS regions (autonomously replicating sequences in yeast) reveals a high AT content, numerous short direct and inverted repeats and the presence of at least one element in each region that is related to the yeast ARS consensus sequence. A/T TTTATPuTTT A/T. These three chloroplast regions share, in addition, two common elements of 10 and 11 bp which may play a role in promoting autonomous replication.

Composite structure of the chloroplast 23 S ribosomal RNA genes of Chlamydomonas reinhardii. Evolutionary and functional implications.

Abstract The chloroplast ribosomal unit of Chlamydomonas reinhardii displays two features which are not shared by other chloroplast ribosomal units. These include the presence of an intron in the 23 S ribosomal RNA gene and of two small genes coding for 3 S and 7 S rRNA in the spacer between the 16 S and 23 S rRNA genes (Rochaix & Malnoe, 1978). Sequencing of the 7 S and 3 S rRNAs as well as their genes and neighbouring regions has shown that: (1) the 7 S and 3 S rRNA genes are 282 and 47 base-pairs long, respectively, and are separated by a 23 base-pair A + T-rich spacer. (2) A sequence microheterogeneity exists within the 3 S RNA genes. (3) The sequences of the 7 S and 3 S rRNAs are homologous to the 5′ termini of prokaryotic and other chloroplast 23 S rRNAs, indicating that the C. reinhardii counterparts of 23 S rRNA have a composite structure. (4) The sequences of the 7 S and 3 S rRNAs are related to that of cytoplasmic 5.8 S rRNA, suggesting that these RNAs may perform similar functions in the ribosome. (5) Partial nucleotide sequence complementarity is observed between the 5′ ends of the 7 S and 3 S RNAs on one hand and the 23 S rRNA sequences which flank the ribosomal intron on the other. These data are compatible with the idea that these small rRNAs may play a role in the processing of the 23 S rRNA precursor.

Localization of 4S RNA genes on the chloroplast genome of Chlamydomonas reinhardii.

SummaryThe genes coding for 4S RNA have been localized on the physical map of the chloroplast genome of Chlamydomonas reinhardii by hybridizing 32P-labelled 4S RNA to EcoRI, BamHI, BgIII and HindIII chloroplast DNA digests and to hybrid plasmids containing EcoRI and BamHI chloroplast DNA fragments. At least 10 EcoRI and 7 BamHI fragments carry sequences coding for 4S RNA. These genes are interspersed throughout the genome. The spacer between the 16S and 23S ribosomal RNA genes, which is repeated twice per chloroplast DNA molecule, codes for at least one 4S RNA, shown to be transcribed from the same strand as the ribosomal RNAs.

Organization of the nuclear ribosomal DNA of Chlamydomonas reinhardii

SummaryHybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×106 d and two SalI ribosomal fragments of 3.8 and 1.5×106 d. The ribosomal DNA (rDNA) units, 5.3×106 d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.