Derivation of a physical map of chloroplast DNA from Nicotiana tabacum by two-dimensional gel and computer-aided restriction analysis

A combined approach was used to derive a detailed physical map of Nicotiana tabacum chloroplast DNA for the restriction enzymes SalI, SmaI, KpnI, and BamHI. Complete maps for the restriction enzymes SalI, SmaI, and KpnI were derived by using two-dimensional agarose gel analysis of fragments obtained by reciprocal double digestion of chloroplast DNA. We have characterized a complete cloned library of N. tabacum chloroplast DNA which contains overlapping restriction fragments resulting from partial digestion by BamHI. With these clones and existing data, we used a novel computer-aided analysis to derive a detailed map for the enzyme BamHI. A comparison and compilation of all published N. tabacum chloroplast DNA restriction maps is presented. Differences between ours and a previously published SmaI and BamHI restriction map are discussed.     

Mapping of rRNA genes in an inverted repeat in Nicotiana tabacum chloroplast DNA.

Nicotiana tabacum chloroplast DNA contains two copies each of 16S and 23S rRNA genes. These genes are located in an inverted order as determined from restriction fragment mapping and Southern hybridization to restriction fragments. The position of these genes on the N. tabacum chloroplast DNA molecule has been established relative to a complete map of SalI and SMaI restriction enzyme cleavage sites.     

The chloroplast genome of Carthamus tinctorius

Summary A physical map of safflower (Carthamus tinctorius L.) chloroplast DNA has been generated using Sall, Pstl, Kpnl and HindIII restriction endonucleases. Southern blots to single and double digests by these enzymes were hybridized with ³²P-dCTP nick-translated Kpnl probes, which were individually isolated from agarose gels. The plastid genome was found to be circular (151 kbp), to contain a repeated sequence of about 25 kbp, and to have small and large single copy regions of approximately 20 and 81 kbp, respectively. Heterologous probes from spinach and Euglena containing psbA, rbcL, atpA or rrnA structural genes were also hybridized with such single and double restriction enzyme digests and mapped on this circular chlorpolast genome. The genetic map was found to be co-linear with that of spinach and many other higher plants.     

Sequence of the genes for tRNACys and tRNAAsp from spinach chloroplasts.

We have determined the map location and primary structure of two fragments of spinach chloroplast DNA which encompass the genes for tRNACysGCA and tRNAAspGUC. Identification of the genes for these two RNA species is based on the sequence of their anticodon triplets and on a comparison of the sequences with those of the equivalent tRNAs from Escherichia coli. Each gene occurs only once on the spinach chloroplast genome and neither contains an intervening sequence. Hybridization of the restriction fragments carrying these genes to chloroplast tRNA showed that both genes are transcribed in vivo.     

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.     

PSIIP680 ChlaAP基因在水稻叶绿体基因组中的定位

本实验总结出一套水稻叶绿体DNA的提取方法,并获得清晰的叶绿体DNA限制性内切酶图谱。Southern杂交结果表明,菠菜PSIIP680ChlaAP基因探针与水稻叶绿体DNA的Pst-1,Pst-14,Pvu-2和Sal-1片段的部分顺序有较高的同源性。根据Hirai和赵衍的水稻叶绿体基因组物理图,可以确定该基因位于紧靠RuBPCaseLS基因,距反向重复区约26kb处。高等植物叶绿体基因组中这种基因排列方式还未见报道。     

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.     

Clone banks of the mung bean, pea and spinach chloroplast genomes

All but one of the PstI restriction fragments from mung bean, pea, and spinach chloroplast DNAs have been stably cloned into pBR322. Large fragments (15-54 kb) were cloned at low efficiencies which decreased with increasing fragment length. However, plasmids containing fragments above 25-30 kb were too unstable to be useful. In particular, pBR322 derivatives containing the largest mung bean and spinach fragments (34 kb and 54 kb, respectively) are extremely unstable and rapidly delete parts of the plasmid sequence. The PstI fragments of mung bean chloroplast DNA which cover the 34-kb PstI fragment have been cloned into pACYC177. After a search of several thousand recombinants we were unable to recover a clone containing a 12.2-kb pea chloroplast PstI fragment and suggest that some property of its sequence may be inimical to the cloning process. The identity of the cloned fragments to native chloroplast DNA restriction fragments is demonstrated by restriction analysis and the ability to construct detailed restriction maps of the mung bean and pea chloroplast genomes.     

Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.     

Construction and mapping of safflower chloroplast DNA recombinants and location of selected gene markers

Summary DNA was isolated and purified from chloroplasts of safflower (Carthamus tinctorius L.), digested with HindIII restriction endonuclease, and ligated into the HindIII site of the plasmid pUC9. Recombinant DNAs were isolated from ampicillin resistant white colonies which grew in the presence of the appropriate indicator, digested with HindIII, and then identified by comparison of agarose gel electrophoretic mobilities. HindIII digests of chloroplast DNA were used as a standard. Such recombinants were radiolabeled and hybridized with Southern blots of PstI, SalI, KpnI, and HindIII single and double digests of safflower chloroplast DNA. A physical map was subsequently generated showing the location of each recombinant on the circular plastid genome. Recombinants containing heterologous chloroplast gene markers from spinach or Euglena were also radiolabeled and mapped. The relative mapping positions of these genes are in good agreement with those which have previously been published for spinach and several other higher plants.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Construction of a SalI/PstI restriction map of spinach chloroplast DNA using low-gelling-temperature-agarose electrophoresis

The restriction endonucleases SalI and PstI cleave circular chloroplast DNA of spinach (Spinacia oleracea) into 12 and 10 fragments, respectively. The sum of the fragment sizes in each of the series is equivalent to the contour length of the molecule (about 95 Md). A physical map was constructed by sequential digestions using low-gelling-temperature agarose to avoid the necessity of extracting the fragments from the gel. The circular DNA molecule of spinach chloroplasts consists of two identical sequences (each about 15 Md) arranged as an inverted repeat separated by two single-copy regions of different sizes (about 52 and 13 Md).     

Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii

The chloroplast DNA of Chlamydomonas reinhardii has been examined by restriction endonuclease analysis. EcoRI, BamHI and BglII produce 30, 17 and 12 fragments, respectively, whose sites have been determined by electron microscopy and by comparative gel electrophoresis. These fragments have been ordered into a circular map which corresponds to a genome size of M_r = 126 × 10⁶. The map was established by comparing the double digests of individual restriction fragments and by hybridizing purified labelled fragments to restriction enzyme digests of chloroplast DNA. The restriction fragments were isolated by molecular cloning or by preparative agarose gel electrophoresis.The two sets of chloroplast ribosomal RNA genes are contained within two inverted repeats of 13 × 10⁶ molecular weight, which are located nearly at opposite sides of the map. In addition, the mapping studies have revealed the presence of short repeated base sequences which are interspersed throughout the chloroplast genome.     

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.     

Extensive rearrangements in the mitochondrial DNA in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana

Summary Mitochondrial DNA (mtDNA) was studied in the leaves of Nicotiana tabacum+Nicotiana knightiana somatic hybrids previously described. Restriction patterns generated by the SalI and BamHI restriction endonucleases were different from both parents in the eight hybrids, and made up of parental and non-parental fragments. Rearrangements in the mtDNAs have been confirmed by DNA-DNA hybridization using, as a probe, labelled 2/12 plasmid DNA which contains the E. coli 16S and 23S rRNA genes. Novel patterns can be explained by new combinations of unaltered parental mtDNA molecules, and by genetic recombination.     

水稻叶绿体DNA克隆片段的分析和rbcL基因在重组质粒上的定位

杂交灿稻(珍汕97B)的叶绿体DNA克隆到pBR 322载体上后,从克隆库中筛选出含核酮糖1.5-二磷酸羧化酶/加氧酶大亚基基因(rbcL)的重组子(19.3kb),用10种限制性内切酶分析了这个重组质粒并制作了完整的物理图谱,rbcL基因被定位在这个物理图谱上。     

Cyanelle DNA from Cyanophora paradoxa

Summary Cyanelles which have been found in few eukaryotic organisms are photosynthetically active organelles which strikingly resemble cyanobacteria. The complexity of the cyanelle genome in Cyanophora paradoxa (127 Kbp) is too low to consider them as independent organisms in a symbiotic relationship. In order to correlate cyanelle genome and gene structure with those of plastid chromosomes of other plants, a circular map of the cyanelle DNA from Cyanophora paradoxa (strain LB555 UTEX) has been constructed using the restriction endonucleases SalI (generating 6 DNA fragments), BamHI (6), SalI (5), XhoI (9), and BglII (19).Besides the rRNA genes (16S, 23S, 5S), genes for 14 proteins have been located on this circular map. Among those are components of several multienzyme complexes involved in photosynthetic electron transport, as well as the large subunit of ribulose-1,5-bisphosphate carboxylase and two ribosomal proteins. All the probes used, were derived from a collection of spinach chloroplast DNA clones. Hybridization experiments showed signals to DNA fragments primarily from the large single-copy region of cyanelle DNA. The arrangement of genes on cyanelle DNA is different from that on spinach chloroplast DNA. However, genes which have been shown to be cotranscribed in spinach chloroplasts are also clustered on cyanelle DNA.Abbreviations Kbp 10³ base pairs - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme     

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.