Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

Microencapsulation of chloroplast particles

Chloroplast and photosystem I particles were encapsulated in small spheres (about 20 μm diameter) with an artificial membrane built up by cross-linking amino groups of protamine with toluenediisocyanate. The artificial membrane was permeable to small substrate and product molecules but not to soluble proteins. Photosystem I activity was retained by the encapsulated chloroplast particles. Washed photosystem I particles were encapsulated with the soluble proteins, ferredoxin, and ferredoxin-NADP oxidoreductase, and the microcapsules photoreduced NADP using ascorbate plus dichlorophenolindophenol as the electron donor. The photosystem I particles were also encapsulated with hydrogenase from Chromatium and a very low rate of photoevolution of hydrogen was obtained. The results show that chloroplast membrane fragments can be encapsulated with soluble proteins that couple transfer reactions to the primary photochemical apparatus.     

History of chloroplast genomics

The presence of chloroplast DNA was established in 1963. With the development of recombinant DNA technologies, chloroplast DNA was selected as one of the first candidates for genome sequencing. The first physical map was reported for maize chloroplasts in 1976. As tobacco has been popular as an experimental system, tobacco chloroplast DNA has been extensively analyzed and the complete nucleotide sequence was established in 1986. This sequencing and the availability of tobacco chloroplast transformation techniques and of in vitro expression systems have formed the basis of an ongoing functional genomics program. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nicotiana chloroplast genome

Summary RuBPCase, the enzyme responsible for carboxylation and oxidation of RuBP in a wide variety of photosynthetic organisms, is the major protein found in the chloroplast. Here we present the first evidence for direct expression in E. coli and B. subtilis of tobacco and Chlamydomonas ct-DNA sequences coding for the LS of RuBPCase as demonstrated by a simple in situ immunoassay.     

Nicotiana chloroplast genome

Summary Using the existing restriction map and probes from wheat and pea ct-DNA, seven protein genes have been localized in the chloroplast genome of N. tabacum. On the clock-like map, the location of each gene is indicated by its time zone: the 15.2 kD polypeptide of the cytochrome b/f complex at 315, cytochrome f at 430, LS of RuBPCase at 450, both and subunits of ATP synthase at or near 500, proton-translocating subunit of ATP synthase at 820, subunit of ATP synthase at 840 and the 32 kD protein at 930. The genome organization of Nicotiana chloroplast DNA is similar to spinach.     

Nicotiana chloroplast genome

Summary A physical map containing six restriction sites of the Nicotiana tabacum chloroplast genome, together with the BamHI maps of N. tabacum, N. otophora and N. knightiana, and the SmaI maps of N. acuminata, N. plumbaginifolia, N. langsdorffii, N. otophora, N. tabacum, N. tomentosiformis and N. knightiana was constructed. In Nicotiana chloroplast genomes, the most frequently observed variations are point mutations. Deletions are also detected. Most of the observed changes are confined to one area of the large single copy region, which is designated as the hot spot. Based on the evidence obtained from Nicotiana chloroplast genomes, an origin of the inverted repeats in this genus is proposed. We suggest that the inverted repeats represent a vestige of what were once two identical, complete chloroplast genomes joined together in a head-to-head and tail-to-tail fashion, and that deletions generated the current chloroplast genome organization.     

Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase

Sequence analysis of a 12,400 base-pair region of the spinach chloroplast genome indicates the presence of three genes encoding subunits of the chloroplast RNA polymerase. These genes are analogous to the rpoBC operon of Escherichia coli, with some significant differences. The first gene, termed rpoB, encodes a 121,000 Mr homologue of the bacterial beta subunit. The second and third genes, termed rpoC1 and rpoC2, encode 78,000 and 154,000 Mr proteins homologous to the N and C-terminal portions, respectively, of the bacterial beta' subunit. RNA mapping analysis indicates that the three genes are cotranscribed, and that a single intron occurs in the rpoC1 gene. No splicing occurs within the rpoC2 gene or between rpoC1 and rpoC2. Furthermore, the data indicate the possibility of an alternative splice acceptor site for the rpoC1 intron that would give rise to a 71,000 Mr gene product. Thus, with the inclusion of the alpha subunit encoded by rpoA at a separate locus, the chloroplast genome is predicted to encode four subunits (respectively called alpha, beta, beta', beta") equivalent to the three subunits of the core enzyme of the E. coli RNA polymerase.     

Gene expression of chloroplast translation elongation factor Tu during maize chloroplast biogenesis

We have examined the expression of a maize nucleartuf gene(tufA) coding for the chloroplast translation elongation factor EF-Tu. Southern analysis revealed that the maize chloroplast EF-Tu was encoded by at least two distinct genes in the nuclear genome. In order to know the effect of light on the expression of thetufA gene during maize chloroplast biogenesis, we have analyzed the steady-state level of thetufA mRNAs by Northern analysis. The steady-state level of thetufA mRNAs was similar in both continuous light- and dark-grown seedlings. The level of thetufA mRNAs also maintained at relatively same level during light-induced greening of etiolated seedlings and all examined developmental stages. These results indicate that the gene expression of the maize chloroplast EF-Tu is rarely light-regulated at it’s mRNA level during chloroplast biogenesis.     

Beta chloroplast genomes: analysis of Fraction I protein and chloroplast DNA variation

Summary The interrelationships of Beta chloroplast genomes have been investigated on the basis of the analysis of Fraction I protein and chloroplast (ct) DNA. Three groups of the chloroplast genomes could be demonstrated by the difference in isoelectric points of the large subunit of Fraction I protein. Restriction enzyme analysis revealed inter- and intra-specific variations among the ctDNAs, which enabled us to detect seven distinct ctDNA types. In Vulgares and Corollinae species, the observed differences were physically mapped taking advantage of the restriction fragment map available for sugar beet (B. vulgaris) ctDNA. The DNA variations were found to result either from gains or losses of restriction sites or from small deletions/ insertions, and most of them were located in the large single-copy region of the genome. Moreover, the ctDNAs from Patellares species are more diverged from those of other Beta taxa. Our results also indicate that there is a close correlation between the chloroplast genome diversity and the accepted taxonomic classification of the species included in this survey.     

Mechanism of chloroplast movement inMougeotia

Summary The single, ribbon-shaped chloroplast in the filamentous green algaMougeotia performs orientational movements with respect to light. The chain of reaction involves phytochrome as the photoreceptor pigment to perceive the light signal differentiated by wavelength and direction, calcium probably to convert the light signal into a chemical message and actomyosin to respond to this message and to move the chloroplast accordingly.Precise reorientation of the chloroplast is proposed to be brought about by a dual function of phytochrome: regulation of the cellular level of calciumand regulation of membrane anchorage sites to actin.The Institute where ProfessorKamiya did early research together with ProfessorKüster.     

Inheritance of chloroplast DNA inPopulus

Summary Restriction fragment length polymorphisms (RFLPs) were used as markers to determine the transmission of chloroplast DNA (cpDNA) in poplar crosses. The plant material studied included individual trees ofPopulus trichocarpa, P. maximowiczii xtrichocarpa, P. maximowiczii xnigra, and offspring from controlled crosses between these trees. RFLPs were identified by direct observation of stained restriction fragments, as well as by molecular hybridization with heterologous cpDNA probes. Analysis of the restriction fragment patterns in the parents and their progeny showed only the patterns of the maternal tree in the progeny, while no paternal type was found. These results provide clear evidence of a maternal mode of chloroplast inheritance in the poplar clones studied.