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Recombinant plasmids containing restriction endonuclease fragments of chloroplast DNA from the alga Euglena gracilis were expressed in an in vitro DNA-dependent transcription-translation system prepared from E. coli . The polypeptides synthesized in this system were radioactively labeled and analyzed by SDS-polyacrylamide gel electrophoresis. Expression of chloroplast DNA fragments cloned in both orientations relative to the plasmid vector and of plasmids containing chloroplast DNA fragments whose nucleotide sequences partially overlap one another permitted the identification of 14 different regions on this organellar genome that have the potential to code for polypeptides.

Cloned chloroplast restriction endonuclease DNA fragments were used as hybridization probes to identify the in vivo transcriptional products of the chloroplast genome from the alga Euglena gracilis. Total cellular RNA was size fractionated by electrophoresis in denaturing gels and transferred to nitrocellulose paper. Individual plasmids containing specific chloroplast DNA fragments were radioactively labeled in vitro and hybridized to the immobilized RNA. The stable RNAs in the chloroplast were identified on the basis of their size and their origin on the chloroplast genome. Several transcripts were shown to be developmentally expressed. Some transcripts showed a possible precursor-product relationship. The rDNA was shown to be transcribed as a large transcript and then processed to the, mature rRNAs.

The nuclear genome of pearl millet has been characterized with respect to its size, buoyant density in CsCl equilibrium density gradients, melting temperature, reassociation kinetics and sequence organization. The genome size is 0.22 pg. The mol percent G + C of the DNA is calculated from the buoyant density and the melting temperature to be 44.9 and 49.7%, respectively. The reassociation kinetics of fragments of DNA 300 nucleotides long reveals three components: a rapidly renaturing fraction composed of highly repeated and/or foldback DNA, middle repetitive DNA and single copy DNA. The single copy DNA consists of 17% of the genome. 80% of the repetitive sequences are at least 5000 nucleotide pairs in length. Thermal denaturation profiles of the repetitive DNA sequences show high T m values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750–1400 nucleotide pairs) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.

A procedure is outlined for purifying DNA from a single plant. A crude organelle pellet consisting of nuclei, chromatin, chloroplasts, and mitochondria is prepared, suspended, and immediately lysed with detergents. The DNA is separated from RNA, protein, and polysaccharides by banding it in CsCl density equilibrium gradients. Ethidium bromide is included in all buffers to act as an inhibitor of DNAase activity. The DNA prepared in this manner can be digested with restriction endonucleases, separated by gel electrophoresis, and used to identify specific genes by hybridization of cloned DNA sequences.

The light-dependent development of the photosynthetic apparatus in the first leaf of the C(4) plant pearl millet (Pennisetum americanum) was monitored by immunologically determining the concentration of phospho-enolpyruvate carboxylase and ribulose 1,5-bisphosphate carboxylase. A competitive enzyme-linked immunosorbent assay procedure using antibodies to the monomeric subunit of phosphoenolpyruvate carboxylase and the large and small subunit of ribulose 1,5-bisphosphate carboxylase was used to quantitate the amounts of these polypeptides in the first leaf of etiolated seedlings and etiolated seedlings exposed to light for varying periods of time. Phosphoenolpyruvate carboxylase was present in etiolated tissue; however, light stimulated its synthesis nearly 23-fold. Maximum accumulation of phosphoenolpyruvate carboxylase occurred approximately 4 days after etiolated plants were placed in the light. Both the large subunit and the small subunit of ribulose 1,5-bisphosphate carboxylase were present in leaves of etiolated seedlings. Light also stimulated the synthesis of both of these polypeptides, but at different rates. In etiolated leaves there was approximately a 3-fold molar excess of the small subunit to large subunit. Exposure of the etiolated leaves to light resulted in the molar ratio of the large subunit to the small subunit increasing to approximately 0.72. These data indicate that the net synthesis of these two polypeptides is not coordinately regulated at all times.

The fraction of chloroplast DNA transcribed at different stages of chloroplast development in Euglena gracilis was measured by RNA-DNA hybridization. Euglena cells were grown in the dark in a heterotrophic medium to stationary phase and then transferred to the light. Chloroplast development was monitored by the increase in the cellular chlorophyll content in the absence of cell division. Total cell RNA was isolated at various stages of chloroplast development, and hybridized in a vast excess to [125I]chloroplast DNA. The fraction of [125I]chloroplast DNA in the form of a duplex was monitored by chromatography on hydroxylapatite columns. The amount of RNA-DNA hybrid in the duplex mixture was determined by correcting for the contribution of DNA-DNA renaturation under the same conditions. The fraction of chloroplast DNA transcribed was calculated by multiplying by two the amount of single-stranded DNA in the form of an RNA-DNA hybrid. Prior to the initiation of chloroplast development (i.e., in dark grown cells) the fraction of chloroplast DNA represented as RNA transcripts in the cell is 0.53. As chloroplast development proceeds, the fraction of the chloroplast DNA transcribed decreases to 0.47. Experiments in which mixtures of various RNA samples were hybridized to the chloroplast DNA indicate that there is a small portion of chloroplast DNA transcribed at later stages of chloroplast development which is not represented as transcripts at the onset of chloroplast development. Melting properties of the RNA-DNA hybrids show that the RNA-DNA duplexes are slightly less stable than renatured [125I]chloroplast DNA.

Chloroplast DNA from Euglena gracilis was separated in an alkaline CsC1 density gradient into a heavy and a light component. The heavy and the light DNA components each contain 18 percent self-complementary nucleotide sequences. Equal masses of the heavy and the light components do not contain equal molar complementary nucleotide sequences. The light DNA component contains sequences complementary to the majority of the chloroplast DNA transcripts. The heavy DNA component contains sequences complementary to chloroplast ribosomal RNA.

The nuclear and chloroplast ribosomal DNAs from Euglena were shown to have specific regions of nucleotide sequence homology. The regions of homology were identified by hybridization of restriction endonuclease DNA fragments of cloned chloroplast and nuclear ribosomal DNAs to one another. The regions of homology between these two ribosomal DNAs were in that part of the genes that code for the 3′ end of the small rRNAs (16S and 19S) and near or at the DNA sequences coding for the 5S RNAs. The nucleotide sequence homology between these regions was estimated to be approximately 94% by the melting point depression of a hybrid formed between the two ribosomal DNAs.

Chloroplast DNA from pearl millet (Pennisetum americanum) was used to construct recombinant plasmids. These plasmids contained 97 kilobase pairs of unique DNA sequences. The chloroplast DNA fragments in these plasmids were mapped with the restriction endonucleases SalI, SphI, XhoI, BglI and HpaI. The technique of overlapping hybridization or chromosome walking was used to orient these DNA fragments on a restriction endonuclease map of the chloroplast genome. The size of the chloroplast DNA from pearl millet was estimated in this fashion to be 127-138 kilobase pairs. Twenty one kilobase pairs of the cloned DNA fragments were represented twice on the genome as inverted repeats. Thus, the recombinant plasmids which were isolated contained approximately 86-93% of the nucleotide sequences in the chloroplast genome of pearl millet. Previously characterized cloned chloroplast DNA sequences from other plants were used as hybridization probes to locate the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase, the β-coupling factor of ATPase and the 32 kilodalton polypeptide of photo system 11 on the restriction endonuclease map of the pearl millet chloroplast genome.

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases Bam HI or Eco RI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases Bam HI, Eco RI, Hin dIII and Hpa I. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a Bam HI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.

Chloroplast DNA from Euglena gracilis was used to construct a partial library of recombinant plasmids representing 45% of the DNA. Each plasmid was radioactively labeled in vitro by nick translation and hybridized in liquid to a vast excess of total cellular RNA isolated either from cells grown continually in the dark or from cells containing developing chloroplasts. The complexity and abundance of the RNA that hybridized to the different chloroplast restriction endonuclease DNA fragments were calculated from the RNA-DNA hybridization saturation values and the pseudo-first-order hybridization rate constants, respectively. The complexity of these transcripts showed little change during chloroplast development. In several cases, the complexity of the RNA was greater than expected for asymmetrical transcription, suggesting the possibility that transcription may be symmetrical in some regions of chloroplast DNA. The abundance of the transcripts ranged from 0.0001% to nearly 10% of the total cellular RNA, and in some cases changed by as much as 5-10-fold during chloroplast development.

The fraction of the chloroplast DNA transcribed in the single celled alga Euglena has been determined by RNA-DNA hybridization. A vast excess of total cell RNA from cells which were rapidly dividing in the light was hybridized in liquid to [125I] — chloroplast DNA, and the resulting duplexes separated on hydroxyapatite columns. The contribution of DNA-DNA duplex formation was determined separately and was used to calculate that portion of the duplex which was actually a RNA-DNA hybrid. Sixteen percent of the single stranded chloroplast DNA forms a duplex with this RNA suggesting that 32 percent of the double stranded DNA molecule is being transcribed into RNA under these conditions of cell growth.

The sequence organization of nuclear DNA in the single-celled alga Euglena gracilis has been studied by a combination of techniques: (1) the comparison of the reassociation kinetics of DNA fragments 300, 2000 and 8100 nucleotides long; (2) the reassociation of 32P-labeled DNA fragments of various lengths with driver fragments 300 nucleotides long; (3) the hyperchromicity of DNA structures formed by the reassociation of repetitive sequences; (4) and the direct measurement of the size of the duplex regions of reassociated repetitive DNA resistant to S1 nuclease. The single copy DNA sequences are approximately 1500 nucleotide pairs long and are interspersed with repetitive DNA sequences. The repetitive DNA, consisting of both highly repetitive and middle repetitive sequences, consists of one fraction of nucleotide sequences (0.67) with an average size of 4900 nucleotide pairs and a second fraction (0.33) with an average size of 1000 nucleotide pairs. 34% of the DNA consists of foldback sequences which are present on 45% of the DNA 4000 nucleotides long.

The chloroplast rDNA genes of pearl millet (Pennisetum americanum) have been cloned and physically mapped. The chloroplast genome of the pearl millet contains two identical rRNA genes located on DNA sequences that are inverted with respect to one another and separated by 12 kb of single-copy DNA. The rRNA genes were positioned on a restriction endonuclease map by using as hybridization probes specific cloned rDNA sequences from the chloroplast DNA of the alga Euglena gracilis. The 16S and 23S rRNA genes were shown to be approx. 2 kb from one another, and the 5S RNA gene is immediately adjacent to the 23S tRNA gene.

A phage λ recombinant library containing Euglena gracilis genomic DNA was screened for nuclear rDNA sequences. A recombinant phage was isolated that contained an 11.5-kb nuclear rDNA sequence. The 11.5-kb insert was mapped with restriction endonucleases and was shown to represent a complete rDNA repeat unit that carried the genes for the 19 S, 25 S, 5.8 S and 5S cytoplasmic rRNAs. The rDNA repeat units per haploid genome are organized in the form of identical tandem repeats.

The reassociation kinetics of Euglena gracilis DNA were carried out to determine the C o t 1 2 of the unique DNA (2000). The C o t 1 2 of the unique DNA and the amount of DNA per cell were used to show that this alga is not polyploid. Three kinetically definable classes of DNA were observed: a highly repetitive fraction, a middle repetitive fraction, and a non-repetitive fraction. Each fraction of DNA was characterized by its melting properties ( T m ).

Transcription of Euglena chloroplast DNA was visualized by autoradiography of hybrids formed between electrophoretically separated EcoRI endonuclease fragments and [32P]-pulse labeled RNA prepared from cells at different stages of chloroplast development. The RNA was hybridized to Southern imprints of EcoRI chloroplast DNA digests. The EcoRI chloroplast DNA fragments were categorized by their ability to hybridize the different pulse labeled RNA's. Some fragments were transcribed: 1. both in the dark and throughout chloroplast development, 2. predominantly in the light, 3. in the dark and for only a brief period of time after transfer of the cells to the light. Other fragments were probably not transcribed.

The number of chloroplast DNA molecules in Euglena gracilis cells was measured by determining the shift in the observed second-order rate constant for the reassociation of 125 I-labeled chloroplast DNA in the presence of unlabeled total cell DNA. Cells grown to stationary phase in the dark contained 217 molecules of chloroplast DNA. Cells grown to stationary phase in the light in either heterotrophic or autotrophic medium contained 590 and 1014 chloroplast DNA molecules, respectively. The observed second-order rate constant for the reassociation of 125 I-labeled chloroplast DNA was not significantly altered in the presence of total cell DNA from a heat-bleached mutant, ZHB, which lacks chloroplast DNA. This evidence suggests that there is less than 0.3 of a chloroplast DNA molecule present in the nucleus of Euglena .

The DNA content of bundle sheath cells and mesophyll protoplasts from the C(4) plant pearl millet (Pennisetum americanum, Tift 23DB) was determined by microspectrophotometry to be 1.8 to 2.3 and 3.2 to 4.0 picograms/nucleus, respectively. Measurement of RNA by ultraviolet spectroscopy indicated that bundle sheath cells contain twice as much RNA as mesophyll cells.