Sequence homologies between Escherichia coli and chloroplast ribosomal DNA as seen by heteroduplex analysis

Circular Vicia faba (broad bean) chloroplast DNA was hybridized to the restriction fragment BamHI B from the DNA of the transducing phage lambda rif^d18, which carries the Escherichia coli ribosomal RNA operon rrnB. Cytochrome spreadings of the heteroduplexes show homologies in the 16 S and 23 S rRNA regions, but none in the spacer. The same lambda rif^d18 fragment was hybridized to the Vicia cpDNA ²SalI fragment 3, which contains the Vicia rBNA operon, resulting in an analogous heteroduplex configuration. Cytochrome spreadings of this heteroduplex in increasing concentrations of formamide reveal regions of incomplete homologies. Heteroduplexes between the E. coli rrnD operon, obtained from the recombinant plasmid pBK8, and circular Vicia cpDNA revealed homologies in the spacer region as well as in the 16 S and 23 S rRNA region. Hybrids between all three types of rDNA and their homologous rRNAs were prepared using the mica adsorption technique. They show that the 23 S, 16 S, and 5 S rRNAs are transcribed from the same strand of Vicia cpDNA. The positions of the rRNAs were measured and compared to the heteroduplex structure. It was observed that the E. coli rrnD operon in the plasmid pBK8 contains two 5 S rRNA sequences near the distal end.     

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.     

tRNA genes in mycobacteria: organization and molecular cloning.

DNAs from nine mycobacteria cleaved with restriction endonucleases were hybridized with cDNA probes synthesized to tRNAs from Mycobacterium tuberculosis and Mycobacterium smegmatis. The tRNA genes are conserved, but their gross genomic organization has diverged in six of the nine species examined. Organisms of the M. tuberculosis H37Ra and H37Rv-M. bovis BCG complex appeared to have identical tRNA gene organization and were indistinguishable from each other. M. tuberculosis and M. smegmatis tRNA-derived cDNA probes hybridized differentially to tRNA-coding DNA segments in five of the species examined, suggesting the existence of qualitatively different tRNA pools in these slow- and fast-growing species. Mycobacterial DNAs hybridized with cDNA synthesized to 23S plus 16S rRNAs from Escherichia coli, and the data suggested that the tRNA genes map close to the rRNA genes. A gene bank of M. tuberculosis H37Rv DNA was constructed, and a recombinant plasmid, pSB2, coding for tRNA(s) and rRNA(s) was partially characterized. Plasmid pSB2 recognized a SalI restriction fragment length polymorphism (RFLP) in M. tuberculosis H37Rv and H37Ra; however, the RFLP is not linked to the tRNA-coding region. To the best of our knowledge, this is the first report of an RFLP which distinguishes the pathogenic strain M. tuberculosis H37Rv from its avirulent derivative H37Ra.     

Restriction endonuclease studies on the chloroplast and mitochondrial DNAs of alfalfa (Medicago sativa L.) protoclones

Summary Alfalfa protoclones were regenerated from the mesophyll protoplasts of two cloned source plants (parents), RS-K1 and RS-K2, initiated from Regen S seed. Because of the high frequency of karyotypic upset previously observed in these plants, chloroplast DNAs (cpDNA) from 23 protoclones and mitochondrial DNAs (mtDNA) from 20 protoclones were examined by restriction endonuclease analysis in order to assess recombination in their cytoplasmic genomes. Seven and four endonucleases were separately used for cpDNA and mtDNA analysis, respectively. Data were consistent with no, or a low frequency of, major sequence rearrangements in either the chloroplast or the mitochondrial genomes as a result of protocloning. However, two types of cpDNA were detected in the 23 protoclones, with only one protoclone possessing the cpDNA type of the cloned parental populations sampled. Possible explanations include a preferential selection during protocloning for one of two parental cpDNA types, an in planta sorting out of cpDNA types in the parental material or both.     

Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome

DNAs from Mycobacterium tuberculosis, M. intracellulare, M. phlei and M. smegmatis were digested by restriction enzymes and hybridized with three probes consisting of the 5' (16S rRNA), the middle (16S and 23S rRNA), and the 3' (23S and 5S rRNA) portions of the Escherichia coli rrnB operon. The resulting hybridization patterns indicate that slow-growing Mycobacteria species (i.e., M. tuberculosis and M. intracellulare), with genome size 3.13 - 4.29 X 10(9) daltons, appear to possess only one rRNA operon, whereas fast-growing species (i.e., M. phlei and M. smegmatis), with genome size 4.30 - 5.20 X 10(9) daltons, appear to possess two rRNA operons.     

Vicia faba chloroplast DNA has only one set of ribosomal RNA genes as shown by partial denaturation mapping and R-loop analysis

Summary Broad-bean (Vicia faba) chloroplast DNA (cpDNA) was isolated and characterized. The intact DNA is circular and has a molecular weight of 79.8x 10⁶ dalton. Electron microscopic analysis of self-annealed intact single-strand circles show that it does not have a large double-stranded inverse repeat as seen in spinach chloroplast DNA. Only one ribosomal RNA gene (one set of 16S and 23S rRNA sequences) was found in preparations of R-loops between the Vicia rRNA and cpDNA circles. A restriction enzyme map for SalI and KpnI was derived by comparing the partial denaturation pattern of the fragments with the pattern of the intact circle. The map was confirmed by gel analysis. The ribosomal RNA gene was localized on the SalI fragment 3b by R-loop analysis. SalI fragment 1a although it contains a G-C rich region did not form R-loops with rRNA. Partial denaturation patterns of spinach cpDNA circles and BglI fragments were determined and from this the position of the fragments mapped. This confirmed the reliability of these methods for the arrangement of restriction enzyme fragments along circular molecules. The structures of the two cpDNAs were compared.     

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.     

Bacteriophage ?29 infection of Bacillus subtilis minicells

Summary Labelled chloroplast rRNAs from Spinacia oleracea were hybridized to restriction endonuclease digests of chloroplast DNA from Oenothera hookeri and Euglena gracilis, to mitochondrial DNA of Acanthamoeba castellanii, and to DNA of the E. coli rrn B operon in the transducing phage lambda rif^d18. The degree of homology is greatest for the 16S rRNA gene. Greater than 90% occurs between the two higher plant genes, 80% homology to the lower plant gene, 60%–70% homology to the bacterial gene, and 20% homology to the mitochondrial gene. The degree of hybridization varied considerably for the 23S and the 5S rRNA genes. Very high homology exists between the two higher plant genes, only about 50% homology for both the Euglena and bacterial genes, and no significant homology for the mitochondrial genes. These results show that any chloroplast (or E. coli) rRNA may be used as a probe to identify rRNA genes in other ctDNAs.Two RNA populations, each enriched for a different ctDNA-encoded mRNA, proved useful in the location of these genes on both higher plant ctDNAs. No significant hybridization was obtained using these probes to the Euglena ctDNA which seems to be too distantly related.Abbreviations Md megadalton, 10⁶ dalton - bp, kbp base pair, kilo base pair - SSC Standard saline citrate, 1 times SSC is 0.15M sodium, chloride, 0.015 M trisodium citrate, pH, 6.8 - mtDNA mitochondrial DNA - ctDNA chloroplast DNA - ctrRNA chloroplast ribosomal RNA     

SEXUALITY AND UNIPARENTAL INHERITANCE OF CHLOROPLAST DNA IN THE ISOGAMOUS GREEN ALGA ULVA COMPRESSA (ULVOPHYCEAE)1

Ulva compressa L. is a heterothallic macroalga considered to be in the early evolutionary stage between isogamy and anisogamy. Two genetic lines of this species, each consisting of gametophytes with opposite mating types, were collected on the coasts of Ehime and Iwate prefectures: MGEC‐1 (mt⁺) and MGEC‐2 (mt^?) from Ehime, and MGEC‐5 (mt⁺) and MGEC‐6 (mt^?) from Iwate. Their relative gamete sizes (i.e., cell volumes) do not correspond to their mating types: MGEC‐6 (19.8 μm³) > MGEC‐1 (18.6 μm³) > MGEC‐5 (17.0 μm³) > MGEC‐2 (10.1 μm³). The pattern of organelle inheritance is an important sexual characteristic in many eukaryotes. We therefore investigated the relationship between gamete size and the inheritance of chloroplast DNA (cpDNA). Polymorphisms between the cpDNA of the two lines were used as markers. We found a 24 bp insertion between psbF and psbL, and the substitution of a StyI site (from C CAAGG to T CAAGG) in the intergenic region between petD and accD. Two interline crosses (MGEC‐1 × MGEC‐6 and MGEC‐2 × MGEC‐5) produced 42 and 38 zygotes, respectively. PCR and PCR–RFLP analyses showed that the cpDNA of the mt⁺ gametes was consistently inherited in both crosses. The cpDNA is inherited from one parent only, and it depends not on gamete size but on being mt⁺. The cpDNA was observed during crossing and in the zygotes 6 h after mating. In 6% of the zygotes, the cpDNA derived from the mt^? gametes disappeared 3–4 h after mating. Preferential digestion of the cpDNA in the zygote’s mt^? gamete may form the basis for uniparental inheritance of cpDNA.     

Characterization of Satellite DNA from Three Marine Dinoflagellates (Dinophyceae): Glenodinium sp. and Two Members of the Toxic Genus, Protogonyaulax

Using CsCl-Hoechst dye or CsCl-ethidium bromide gradients, satellite and nuclear DNAs were separated and characterized in three marine dinoflagellates: Glenodinium sp., and two toxic dinoflagellates, Protogonyaulax tamarensis and Protogonyaulax catenella. In all three dinoflagellates, the lowest density fraction, satellite DNA₁, hybridized to chloroplast genes derived from terrestrial plants and/or other algae. Dinoflagellate chloroplast DNAs exhibited molecular sizes of 114 to 125 kilobase pairs, which is consistent with plastid sizes determined for other chromophytic algae (120-150 kilobase pairs). Mitochondrial DNA was not resolved from nuclear DNA in this system. Two additional satellite DNAs, satellite DNA₂ and satellite DNA₃, recovered from P. tamarensis and P. catenella were similar to one another, both within and between species, when characterized by restriction enzyme analysis. These satellites were 85 to 95 kilobase pairs in size, and exhibited restriction fragments that hybridized to yeast nuclear ribosomal RNA genes. Restriction enzyme analyses and DNA hybridization studies of cpDNA document that the two Protogonyaulax isolates are not evolutionarily identical.     

Chloroplast DNA variability in the genus Helianthus: restriction analysis and S1 nuclease mapping of DNA-DNA heteroduplexes

Chloroplast DNA (cpDNA) from 36 wild species of the genus Helianthus has been analysed with three restriction endonucleases (Bam HI, Hind III and Sst I). Out of the 71 restriction sites described on the reference cpDNA (sunflower cpDNA), three insertions/deletions and seven site modifications were detected during the survey of the other cpDNAs.Since restriction mapping showed only a very limited fraction of the DNA variability, we chose to adapt the S1 nuclease mapping technique to detect fine variations between chloroplast genomes. For this purpose, DNA-DNA heteroduplexes obtained between sunflower and wild-species DNAs were digested by S1 nuclease and the resulting mismatches were detected by classical endonuclease restriction and hybridization methods. The S1 nuclease mapping results were confirmed by sequencing one S1 nuclease-sensitive region detected between cultivated sunflower and two perennial wild-type species.As a result of these analyses, it appeared that the combination of restriction mapping and S1 nuclease mapping might be helpful to differentiate taxonomically close cytoplasms.     

Mapping of the ribosomal RNA genes on spinach chloroplast DNA.

Spinach chloroplast ribosomal RNAs have been hybridized to restriction endonuclease fragments of spinach chloroplast DNA. All three RNA species (23S, 16S and 5S) hybridized to a single large fragment when the DNA was digested with either Sall or Pstl. Hybridization of 23S RNA to fragments produced by Smal yielded two radioactive bands which corresponded to the bi-molar 2.5 X 10(6) and 1.15 X 10(6) Mr fragments. 16S RNA also hybridized to two, bi-molar Smal fragments (3.4 X 10(6) and 2.5 X 10(6) Mr) and 5S RNA hybridized to the 1.15 X 10(6) Mr bi-molar Smal fragment. The 23S RNA and 16S RNA cistrons were each also shown to contain a single EcoRI site. From the data it was possible to conclude that the ribosomal RNA genes are located on the inverted repeat region of the spinach chloroplast DNA restriction map [1,2], that the sequence of the cistrons is 16S - 23S - 5S and that the size of the spacer between the 16S and 23S RNA cistrons is approximately 0.90 X 10(6) Mr.     

Sequence studies on the soybean chloroplast 16S–23S rDNA spacer region

The sequence of the ribosomal spacer region of soybean chloroplast DNA including the 3 end of the 16S rRNA gene, the tRNA^Ala and tRNA^Ile genes (but not their introns), the three intergenic regions and the 5 end of the 23S rRNA gene, has been determined. This sequence has been compared to corresponding regions of other angiosperm chloroplast DNAs. Secondary structure models are proposed for the entirety of the intergenic regions a, b and c and for the flanking rRNA regions. A model for a common secondary structure of the ribosomal spacer intergenic regions from chloroplasts of higher plants is proposed, which is supported by comparative evidence.     

Transfer RNA genes associated with the 16S and 23S rRNA genes of Euglena chloroplast DNA

Hybridization studies of Euglena chloroplast ¹²⁵I-labeled tRNAs to restriction fragments of Euglena chloroplast DNA have shown that the spacer between the 16S and 23S rRNA genes, in two and possibly all three of the ribosomal DNA units, contains genes for tRNA^Ile and tRNA^Ala, whereas a tRNA gene (for either tRNA^Trp or tRNA^Glu) is located before probably all four 16S rRNA genes present on the chloroplast DNA molecule.     

DNA EXTRACTION METHODS FOR KELP (LAMINARIALES) TISSUE1

Newly adapted extraction methods for harvesting total cellular DNA from kelp (Laminariales; Phaeophyta) exploit the life-history stages of these heteromorphic algae. Earlier techniques, developed primarily for chloroplast DNA extraction, were time consuming and labor intensive and required large quantities of fresh sporophyte tissue. In contrast, the new methods expedite DNA extraction by employing dried sporophyte laminae or fresh gametophyte filaments and meiospores. The current methods require less tissue and procedural manipulation, reducing the time and labor involved in DNA extraction. Additionally, the current methods were successfully employed to extract DNA from herbarium specimens up to 22 years old. Total cellular DNA yields were 12.8 and 26.4 μg:g^?1 from dried laminae,: 1.9 and 11 μg from small volumes of concentrated gametophyte filaments (25 μL), and 43 to 54.5 μg from pellets of isolated meiospores (50 μL). Following purification on Sepharose columns, DNA from all three sources was sufficiently pure for molecular applications, including restriction endonuclease digestion, Southern blot-hybridizations, and amplification via the polymerase chain reaction.     

Chloroplast DNA differences between two species of Oenothera subsection Munzia: location in the chloroplast genome and relevance to possible interactions between nuclear and plastid genomes

Summary The chloroplast DNAs (cpDNAs) of Oenothera berteriana and Oe. odorata (subsection Munzia) were examined by restriction endonuclease analysis with Sal I, Pvu II, Kpn I, Pst I, Hind III, and Bam HI. The fragment patterns show that these cpDNAs have all 133 restriction sites in common as well as a lot of individual bands. Nevertheless the cpDNAs of the two species can be distinguished by distinct differences in size between a small number of fragments. The 42 cleavage sites produced by Sal I, Pvu II and Kpn I were mapped on the circular cpDNAs. This was achieved by an approach which combined experimental and mathematical procedures. The overall serial order of the fragments was found to be the same for both cpDNAs. The size differences of individual fragments in the Sal I, Pvu II and Kpn I patterns between Oe. berteriana and Oe. odorata cpDNA are located within five regions scattered along the plastid chromosome. Two of these regions have been localized in the larger and one in the smaller of the two single-copy parts of the cpDNA molecule. The remaining two overlap the borders between the large single-copy and each of the duplicated parts of the molecule. The positions of distinct restriction sites are altered among the two Oenothera plastome DNAs by 0.02–0.4 MDa (30–600 base pairs). These alterations probably result from insertions/deletions.Abbreviations cpDNA chloroplast, plastid DNA - Oe. Oenothera - MDa Megadalton - rRNA, rDNA ribosomal RNA, DNA Dedicated to Professor Berthold Schwemmle, Tübingen, on the occasion of his 60th birthday     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

Peculiar feature of the organization of rRNA genes of the Chlorella chloroplast DNA.

The organization of a cloned rRNA gene cluster from Chlorella ellipsoidea chloroplast DNA (cpDNA) has been analyzed. Southern hybridization experiments with labelled chloroplast rRNAs as probes revealed an extraordinarily large size of the 16S-23S rRNA spacer region, ca. 4.8 kbp, almost twice as large as those of most higher plants. The nucleotide sequence determined on this region has shown that: (1) The tRNAIle gene locating in this region is similar to those of higher plant chloroplasts, blue-green algae and E. coli but does not contain any introns in contrast to higher plant chloroplasts. (2) The tRNAAla gene is absent from this region. (3) There are four open reading frames (ORFs) coding for 55, 102, 107 and 110 amino acids, respectively. (4) A few sets of unique sequence were found repeatedly in this region. (5) The 23S rRNA gene is coded on the opposite strand in the reverse order. This arrangement of the 16S-23S rRNA region of Chlorella cpDNA is quite different from any of those reported so far for various organisms.     

Chloroplast DNA inheritance in Populus

Summary The inheritance of chloroplast (cp) DNA was examined in F₁ hybrid progenies of two Populus deltoides intraspecific controlled crosses and three P. deltoides × P. nigra and two P. deltoides × P. maximowiczii interspecific controlled crosses by restriction fragment analysis. Southern blots of restriction digests of parental and progeny DNAs were hybridized to cloned cpDNA fragments of Petunia hybrida. Sixteen enzymes and five heterologous cpDNA probes were used to screen restriction fragment polymorphisms among the parents. The mode of cpDNA inheritance was demonstrated in progenies of P. deltoides × P. nigra crosses with 26 restriction fragment polymorphisms of cpDNA differentiating P. deltoides from P. nigra, as revealed by 12 enzyme-probe combinations, and in progenies of P. deltoides × P. maximowiczii crosses with 12 restriction fragment polymorphisms separating P. deltoides from P. maximowiczii, as revealed by 7 restriction enzyme-probe combinations. In all cases, F₁ offspring of P. deltoides × P. nigra and P. deltoides × P. maximowiczii crosses had cpDNA restriction fragments of only their maternal P. deltoides parent. The results clearly demonstrated uniparental-maternal inheritance of the chloroplast genome in interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii. Intraspecific P. deltoides hybrids also had the same cpDNA restriction fragments as their maternal parent. Maternal inheritance of the chloroplast genome in Populus is in agreement with what has been observed for most other angiosperms.