DNA and RNA Levels in Bundle Sheath and Mesophyll Cells of Pearl Millett (Pennisetum americanum).

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.

Immunological determination of phosphoenolpyruvate carboxylase and the large and small subunits of ribulose 1,5-bisphosphate carboxylase in leaves of the c(4) plant pearl millet.

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.

Chloroplast DNA variation in pearl millet and related species.

The evolution of specific regions of the chloroplast genome was studied in five grass species in the genus Pennisetum, including pearl millet, and one species from a related genus (Cenchrus). Three different regions of the chloroplast DNA were investigated. The first region included a 12-kilobase pair (kbp) EcoRI fragment containing the 23S, 16S and 5S ribosomal RNA genes, which is part of a larger duplicated region of reverse orientation. The second region was contained in a 21-kbp Sa/I fragment, which spans the short single-copy sequence separating the two reverse repeat structures and which overlaps the duplicated copies of the 12-kbp Eco RI fragment. The third region was a 6-kbp EcoRI fragment located in the large single-copy region of the chloroplast genome. Together these regions account for slightly less than 25% of the chloroplast genome. Each of these DNA fragments was cloned and used as hybridization probes to determine the distribution of homologous DNA fragments generated by various restriction endonuclease digests.-A survey of 12 geographically diverse collections of pearl millet showed no indication of chloroplast DNA sequence polymorphism, despite moderate levels of nuclear-encoded enzyme polymorphism. Interspecific and intergeneric differences were found for restriction endonuclease sites in both the small and the large single-copy regions of the chloroplast genome. The reverse repeat structure showed identical restriction site distributions in all materials surveyed. These results suggest that the reverse repeat region is differentially conserved during the evolution of the chloroplast genome.

A restriction endonuclease map of the chloroplast genome of pearl millet.

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 ß-coupling factor of ATPase and the 32 kilodalton polypeptide of photo system 11 on the restriction endonuclease map of the pearl millet chloroplast genome.

In vitro coupled transcription-translation of linear DNA fragments in a lysate derived from a recB rna pnp strain of Escherichia coli.

A recB21 derivative (CLB7) of an Escherichia coli rna-19 pnp-7 strain (PR7) was constructed for use in examining the in vitro coupled transcription-translation of linear DNA. The expression of linearized DNAs in CLB7 (recB21 rna-19 pnp-7) lysates was enhanced significantly when compared with expression of the same DNAs in lysates prepared from the PR7 or the original recB21 (CF300) strains. In addition, the endogenous incorporation of [35S]methionine into protein was considerably reduced in CLB7 lysates relative to lysates derived from the original recB21 strain.

In vivo transcriptional products of the chloroplast DNA of Euglena gracilis.

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.

Purification of total cellular DNA from a single plant.

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.

Nucleotide Sequence Homology Exists between the Chloroplast and Nuclear Ribosomal DNAs of Euglena gracilis.

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.

A restriction map of the ribosomal RNA genes and the short single-copy DNA sequence of the pearl millet chloroplast genome.

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.

Characterization of the nuclear ribosomal DNA of Euglena gracilis.

A phage lambda 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 19S, 25S, 5.8 S and 5 S cytoplasmic rRNAs. The 2000 rDNA repeat units per haploid genome are organized in the form of identical tandem repeats.

Complexity and abundance of ribonucleic acid transcribed from restriction endonuclease fragments of Euglena chloroplast deoxyribonucleic acid during chloroplast development.

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.

Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum.

Thirteen strains of a strict anaerobic, extreme thermophilic bacterium were isolated from soil samples of moderate temperature, from a sewage plant in Georgia, and from hot springs in Utah and Wyoming. They were identified as strains of Clostridium thermohydrosulfuricum. The guanosine + cytosine content (moles percent) was 37.6 (determined by buoyant density) and 34.1 (determined by melting temperature). All strains required a factor present in yeast extract or tryptone growth. Growth characteristics were as follows: a pH range of 5 to 9, with the optimum between 6.9 to 7.5, in a temperature range of 40 to 78 degrees C, with the optimum at 68 degrees C. The doubling time, when grown on glucose at temperature and pH optima, was 1.2 h. The main products of glucose fermentation were ethanol, lactate, acetate, CO2, and H2. The fermentation was inhibited by H2. Formation of spores occurred easily on glucose-agar medium or when cultures growing at temperatures above 65 degrees C were allowed to cool to temperature below 55 degrees C. C. thermohydrosulfuricum occurs widely distributed in the natural environment.

DNA sequence organization in the alga Euglena gracilis.

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.

Characterization of the nuclear genome of pearl millet.

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 Tm values implying a high degree of sequence homogeneity. About half of the single copy DNA is short (750--1400 nucleotide paris) and interspersed with long repetitive DNA sequences. The remainder of the single copy sequences vary in size from 1400 to 8600 nucleotide pairs.

Influence of growth conditions upon the number of chloroplast DNA molecules in Euglena gracilis.

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.