Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.     

Construction of fowlpox virus vectors with intergenic insertions: expression of the beta-galactosidase gene and the measles virus fusion gene.

A DNA fragment from fowlpox virus cloned on a plasmid vector was modified to contain foreign DNA inserts within an intergenic region. In a first step, a 32-base-pair intergenic region from the fowlpox virus genome corresponding to the position of the thymidine kinase locus in the vaccinia virus genome was enlarged to 55 base pairs by site-directed mutagenesis. A unique restriction endonuclease site introduced upstream of the intergenic region was then used to insert various foreign DNA fragments. The lacZ gene encoding beta-galactosidase and the measles virus gene encoding the fusion protein were positioned downstream of two vaccinia virus p7.5 promoter elements in either a direct repeat or inverted repeat orientation. Foreign DNA inserts contained within the fowlpox virus sequence were transferred to the viral genome by homologous recombination occurring in cells infected with a fowlpox virus temperature-sensitive mutant and transfected with both wild-type viral DNA and plasmid DNA. Recombinant viruses were selected for the expression of beta-galactosidase activity by screening for blue plaques in the presence of a chromogenic substrate. Stable recombinants expressing both the lacZ gene and the unselected measles gene were obtained when the p7.5 promoter was present as an inverted repeat. However, when the p7.5 promoter was in the direct repeat orientation, viral recombinants which initially expressed both gene inserts readily deleted the lacZ gene flanked by the promoter repeat. The methods described enable precise insertion and deletion of foreign genes in the fowlpox virus genome and could be applied to other intergenic regions of the same virus as well as other poxviruses.     

Rice chloroplast DNA: a physical map and the location of the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase and the 32 KD photosystem II reaction center protein

Summary By homogenizing rice leaves in liquid nitrogen, it was possible to isolate intact chloroplasts and, subsequently, pure rice chloroplast DNA from the purified chloroplasts. The DNA was digested by several restriction enzymes and fragments were fractionated by agarose gel electrophoresis. The sum of the fragment sizes generated by the restriction enzymes showed that the total length of the DNA is 130 kb. A circular physical map of fragments, generated by digestion with SalI, PstI, and PvuII, has been constructed. The circular DNA contains two inverted repeats of about 20 kb separated by a large, single copy region of about 75 kb and a short, single copy region of about 15 kb. The location of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase (Fraction I protein) and the 32 KD photosystem II reaction center gene were determined by using as probes tobacco chloroplast DNAs containing these genes. Rice chloroplast DNA differs from chloroplast DNAs of wheat and corn as well as from dicot chloroplast DNAs by having the 32 KD gene located 20 kb removed from the end of an inverted repeat instead of close to the end, as in other plants.     

Chloroplast DNA replication in vitro: site-specific initiation from preferred templates.

An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17' (2470 bp) and Eco RI x (1368 bp) have been shown to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194-198]. Analysis of replicative intermediates indicates that although the template activity of the recombinant plasmid pZmcBam 17' is substantially greater than that of the pZmcEco x, replication in both cases originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion of maize chloroplast BamHI fragment 17' is not more active because it contains additional origins for replication. The overlapping region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455 bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA insert, a region that includes the 3' terminal part of the psbA gene.     

Maize chloroplast DNA encodes a protein sequence homologous to the bacterial ribosome assembly protein S4.

A cloned restriction fragment of maize chloroplast DNA (Bam H1 fragment 5) is shown to contain an open reading frame which encodes a basic protein of 201 amino acid residues with 40-50% sequence homology to E. coli ribosomal protein S4. Based on the experimentally determined sequence homology between the highly conserved bacterial ribosomal protein L12 and its chloroplast homologue (Bartsch M., Kimura, M. and Subramanian, A.R. (1982) Proc. Natl. Acad. Sci. USA 79, 6871), we conclude that this reading frame represents the maize chloroplast S4 gene. The nucleotide sequence of a 1100 base pair DNA segment containing the putative gene is presented.     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

Mapping of a vaccinia host range sequence by insertion into the viral thymidine kinase gene.

A vaccinia virus mutant deleted of ca. 18 kilobase pairs at the left-hand end of the genome is unable to multiply on many human cell lines. To determine whether all or some of the deleted sequences were responsible for the host range property, the corresponding region from wild-type DNA was cloned in three pieces into a vaccinia transplacement vector containing the thymidine kinase gene on the HindIII J fragment. The next step was to transfer these pieces to the genome of the host range deletion mutant by in vivo homologous recombination around the thymidine kinase locus. Transfer of one 5.2-kilobase-pair EcoRI fragment was found to restore a wild-type phenotype on the host range mutant, thus demonstrating that only a small portion of the 18-kilobase-pair deletion contains the host range function(s). This result also illustrates that the method initially devised for inserting foreign genes into vaccinia virus DNA is useful for studies of the vaccinia genome.     

Isolation of the structural gene encoding a mutant form of Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate. Identification of an amino acid substitution in the mutant

The structural gene encoding a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate (Fru-P2) was isolated from total E. coli PpcI genomic DNA. This mutant gene is located on a 4.4-kilobase SalI DNA fragment which, when ligated to SalI-digested pBR322, resulted in the generation of the plasmid pFS16. Detailed restriction mapping of the wild-type and mutant genes for phosphoenolpyruvate carboxylase revealed the presence of a ClaI restriction site at position 563 of the mutant gene only. This ClaI site is located on a 289 PvuII/DdeI fragment which codes for amino acid residues 174-270 of the phosphoenolpyruvate carboxylase enzyme. When this portion of the mutant gene is present in chimeras of the wild-type and mutant genes, the phosphoenolpyruvate carboxylase produced cannot be activated by Fru-P2. The mutation resulting in the generation of the ClaI site in the mutant gene has also resulted in an amino acid substitution at residue 188; threonine in the wild-type enzyme has been replaced by isoleucine in the mutant enzyme. Comparison of the nucleotide sequence of this 289-base pair PvuII/DdeI region of the mutant gene with its homologous region in the wild-type gene verified that this mutation, which resulted in the generation of the ClaI site, is the only change that has occurred on this 289-base pair fragment of the mutant gene, and thus the amino acid replacement of threonine by isoleucine is the only change that could be linked to the inability of the mutant enzyme to be activated by Fru-P2.     

The replication origin of proplastid DNA in cultured cells of tobacco

Summary When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.     

Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line.

It has been reported that SV40-transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) produce a super T antigen of 115,000 M. This super T antigen is entirely SV40 coded and is synthesized by translation of an elongated form of SV40 early mRNA (May, E., Kress, M. Daya-Grosjean, L., Monier, R. and May, P. (1981) J. Virol., 37, 24-35). The results reported here show that there is only one independent insertion of viral DNA in the cellular genome of subclone 7 cells. When DNA from subclone 7 cells was cleaved with Bam HI endonuclease two distinct SV40 sequence containing fragments were generated with sizes of 5 Kb and 10 Kb, respectively. Two recombinant cosmids were constructed by insertion of the 5 Kb and 10 Kb fragments, respectively, into cosmid pHC 79. Using restriction map analysis and nucleotide sequencing, we showed that the 5 Kb fragment actually contained the complete sequence of a gene encoding super T antigen. As compared to the normal SV40 early gene, the sequence of super T gene showed the following rearrangements: (i) The segment between nucleotides 4116 - 3544 was duplicated in a direct order and (ii) these two copies of 573 nucleotide sequence were separated by a 93 nucleotide tract which was a nearly perfect inverted repeat of the segment located between nucleotides 4868 and 4776 (nucleotide numbering used here = Weissmann number +17).     

Analysis of two gene regions involved in the expression of the imipenem-specific, outer membrane porin protein OprD of Pseudomonas aeruginosa

A Tn501 mutant of Pseudomonas aeruginosa resistant to imipenem and lacking the imipenem-specific outer membrane porin protein OprD was isolated. The mutation could be complemented to imipenem susceptibility and OprD-sufficiency by a cloned 6-kb EcoRI-PstI fragment of DNA from the region of chromosome of the wild-type strain surrounding the site of Tn501 insertion. However, this fragment did not contain the oprD structural gene as judged by its inability to hybridize with an oligonucleotide corresponding to the N-terminal amino acid sequence of OprD. DNA sequencing of 3.9 kb of the region surrounding the Tn501 insertion site revealed three large open reading frames, one of which would be interrupted by the Tn501 insertion in the mutant. This latter open reading frame, named opdE (for putative regulator of oprD expression), predicted a hydrophobic protein of M(r) 41,592. Using the above-mentioned oligonucleotide, the oprD structural gene was cloned and expressed in Escherichia coli on a 2.1-kb Bam HI-KpnI fragment. DNA sequencing predicted a 420 amino acid mature OprD protein with a 23 amino acid signal sequence.     

Chloroplast transfer RNAs and tRNA genes of wheat

Fractionation (by two-dimensional polyacrylamide gel electrophoresis) of total tRNA from wheat chloroplasts yields about 33 RNA spots. Of these, 30 have been identified by aminoacylation as containing tRNAs specific for 17 amino acids. Hybridization of labeled individual tRNAs to cloned chloroplast DNA fragments has revealed the location of at least nine pairs of tRNA genes in the segments of the inverted repeat, at least twelve tRNA genes in the large single copy region and one tRNA gene in the small single copy region. A comparison of this wheat chloroplast tRNA gene map to that of maize and of other higher plants suggests that gene rearrangements have occurred during evolution, even within cereal chloroplast DNA. These rearrangements have taken place within the inverted repeat, within the large single copy region and between the inverted repeat and the large single copy region.     

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.     

Mapping of transfer RNA genes on tobacco chloroplast DNA

Tobacco chloroplast tRNAs have been purified by two-dimensional polyacrylamide gel electrophoresis, identified by aminoacylation, labelled at their 3-end and hybridized to tobacco chloroplast DNA restriction fragments, in order to establish a tRNA gene map. These hybridization studies have revealed the localization of at least seven genes in each inverted repeat region, a minimum of 22 tRNA genes in the large single copy region and one tRNA gene in the small single copy region. Comparison of the tobacco chloroplast tRNA gene map to that of maize shows many similarities, but also some differences suggesting that DNA sequence rearrangements have occurred in the chloroplast genome during evolution.