Conserved gene clusters in the highly rearranged chloroplast genomes of Chlamydomonas moewusii and Chlamydomonas reinhardtii

We have extended to about 75 the number of genes mapped on the Chlamydomonas moewusii and Chlamydomonas reinhardtii chloroplast DNAs (cpDNAs) by partial sequencing of the very closely related C. eugametos and C. moewusii cpDNAs and by hybridizations with Chlamydomonas chloroplast gene-specific sequences. Only four of these genes (tscA and three reading frames) have not been identified in any other algal cpDNAs and thus may be specific to Chlamydomonas. Although the C. moewusii and C. reinhardtii cpDNAs differ by complex sequence rearrangements, 38 genes scattered throughout the genome define 12 conserved clusters of closely linked loci. Aside from the rRNA operon, four of these gene clusters share similarity to evolutionarily primitive operons found in other cpDNAs, representing in fact remnants of these operons. Our results thus indicate that most of the ancestral bacterial operons that characterize the chloroplast genome organization of land plants and early-diverging photosynthetic eukaryotes have been disrupted before the emergence of the polyphyletic genus Chlamydomonas. All gene rearrangements between the C. moewusii and C. reinhardtii cpDNAs, with the exception of those accounting for the relocations of atpA, psbI and rbcL, occurred within corresponding regions of the genome. One of these rearrangements seems to have led to disruption of the ancestral region containing rpl23, rpl2, rps19, rpl16, rpl14, rpl5, rps8 and the psaA exon 1. This gene cluster, which bears striking similarity to the Escherichia coli S10 and spc operons, spans a continuous DNA segment in C. reinhardtii, while it maps to two separate fragments in C. moewusii.     

Isolation,physical map and gene map of mitochondrial DNA from the cryptomonad Pyrenomonas salina

Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.     

Application of physical and genetic map of Rhizobium leguminosarum bv. trifolii TA1 to comparison of three closely related rhizobial genomes

A combined physical and genetic map of Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) genome was constructed and used in comparison of chromosomal organization with the closely related R. leguminosarum bv. viciae 3841 (Rlv) and Rhizobium etli CNF42 (Rhe). This approach allowed evaluation of chromosome and genome plasticity and provided important insights into R. leguminosarum lineage diversity. MssI, SmiI, PacI, and I-CeuI restriction endonucleases were chosen for the analysis, generating fragments with suitable size distributions for RtTA1 genome mapping. The fragments were assembled into a physical map using a combination of complementary methods, including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. About 100 genetic markers were located on the RtTA1 restriction map. Comparison of genetic maps of RtTA1, Rlv, and Rhe revealed extensive chromosomal colinearity despite differences in the physical maps. The comparison provides bases for comprehensive analysis of the evolution of R. leguminosarum genome, indicating that, at least on the chromosomal level, no major rearrangements had occurred after the evolutionary divergence of R. leguminosarum biovars. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

The mitochondrial genome of an exsymbiotic Chlorella-like green alga

Mitochondrial (mt) DNA from the unicellular, exsymbiotic Chlorella-like green alga, strain Nla was isolated and cloned. The mtDNA has a buoyant density of 1.692 g/ml in CsCl and an apparent G/C base composition of 32.5%. The genome contains approximately 76 kbp of DNA based on restriction fragment summation and electron microscopic measurements. A map of restriction endonuclease sites using Sst I, Bam I, Sal I and Xho I was generated. The genome maps as a circular molecule and appears as such under the electron microscope. Eight genes were assigned to the map by hybridization to specific restriction fragments using heterologous mt-encoded specific probes. These include the genes for subunits 6, 9, and alpha of the F₀-F₁ ATPase complex, the large and small subunit rRNAs, cytochrome oxidase subunits I and II, and apocytochrome b.     

Physical and genetic map of the Methanobacterium wolfei genome and its comparison with the updated genomic map of Methanobacterium thermoautotrophicum Marburg

A physical and genetic map of the chromosome of Methanobacterium wolfei was constructed by using pulsed-field gel electrophoresis of restriction fragments generated by digestion with NotI and NheI. The chromosome was found to be circular and 1,729 kb in size. Twenty-eight genes were mapped to specific restriction enzyme fragments by performing hybridization experiments with gene probes from various Methanobacterium strains. The genomic map obtained was compared with the updated genomic map of Methanobacterium thermoautotrophicum Marburg. In spite of major restriction pattern dissimilarities, the overall genetic organization seemed to be conserved between the genomes of the two strains. In addition, the two rRNA operons of strain Marburg were precisely mapped on the chromosome, and it was shown that they are transcribed in the same direction.     

Chloroplast genome organization of bromegrass,Bromus inermis Leyss

Summary A physical map of the Bromus inermis chloroplast genome was constructed using heterologous probes of barley and wheat chloroplast DNA (cpDNA) to locate restriction sites. The map was aligned from data obtained from filter hybridization experiments on single and double enzyme digests. Cleavage sites for the enzymes PstI, SalI, KpnI, XhoI and PvuII were mapped. The chloroplast genome of B. inermis is similar in physical organization to that of other grasses. The circular cpDNA molecule of B. inermis has the typical small (12.8 kbp) and large (81.3 kbp) single-copy regions separated by a pair of inverted repeat (21 kbp) regions. The cpDNA molecule of B. inermis is collinear in sequence to that of wheat, rye, barley and oats. No structural rearrangements or major deletions were observed, indicating that the cpDNA of Bromus is a useful tool in phylogenetic studies.     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

Physical and Genetic Map of the Genome of Buchnera, the Primary Endosymbiont of the Pea Aphid Acyrthosiphon pisum

Abstract The genome of Buchnera, an endosymbiotic bacterium of the pea aphid Acyrthosiphon pisum, was characterized by pulse-field gel electrophoresis (PFGE) as a circular DNA molecule of 657 kb. The enzymes I-CeuI, CpoI, ApaI, SmaI, NaeI, SacII, MluI, FspI, and NruI were used to cleave the DNA of Buchnera into fragments of suitable size for PFGE analysis. A physical map of the Buchnera genome, including restriction fragments from seven of these enzymes, was constructed using double cutting, partial digestion, and hybridization with linking fragments, and 29 genes and operons were localized on the map. In addition, the genomic map of Buchnera was compared with those of Escherichia coli and Haemophilus influenzae. The gene order in Buchnera is more similar to that of E. coli than to H. influenzae. The dramatic shrinkage of the Buchnera genome compared with those of other members of the closely related Enterobacteriaceae family is discussed in terms of evolution under the influence of the intracellular symbiotic association. Received: 20 May 1998 / Accepted: 1 July 1998     

Physical map and gene organization of the mitochondrial genome from the unicellular green alga Platymonas (Tetraselmis) subcordiformis (Prasinophyceae)

the entire mitochondrial genome (mt genome) of the unicellular green alga Platymonas subcordiformis (synonym Tetraselmis subcordiformis; Prasinophyceae) was cloned and a physical map for the four restriction enzymes Hind III, Eco RI, Bgl II and Xba I was constructed. The mt genome of P. subcordiformis is a 42.8 kb circular molecule, coding for at least 23 genes. Hybridization and sequence analysis revealed the presence of a ca. 1.5 kb inverted repeat on the mt genome of P. subcordiformis. Phylogenetic analyses based on sequences of several coxI genes were carried out. Our data indicate that mitochondria from P. subcordiformis and from land plants form a natural, monophyletic group.     

Genomic organization of lactic acid bacteria

Current knowledge of the genomes of the lactic acid bacteria, Lactococcus lactis and Streptococcus thermophilus, and members of the genera Lactobacillus, Leuconostoc, Pediococcus and Carnobacterium is reviewed. The genomes contain a chromosome within the size range of 1.8 to 3.4 Mbp. Plasmids are common in Lactococcus lactis (most strains carry 4–7 different plasmids), some of the lactobacilli and pediococci, but they are not frequently present in S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus or the intestinal lactobacilli. Five IS elements have been found in L. lactis and most strains carry multiple copies of at least two of them; some strains also carry a 68-kbp conjugative transposon. IS elements have been found in the genera Lactobacillus and Leuconostoc, but not in S. thermophilus. Prophages are also a normal component of the L. lactis genome and lysogeny is common in the lactobacilli, however it appears to be rare in S. thermophilus. Physical and genetic maps for two L. lactis subsp. lactis strains, two L. lactis subsp. cremoris strains and S. thermophilus A054 have been constructed and each reveals the presence of six rrn operons clustered in less than 40% of the chromosome. The L. lactis subsp. cremoris MG1363 map contains 115 genetic loci and the S. thermophilus map has 35. The maps indicate significant plasticity in the L. lactis subsp. cremoris chromosome in the form of a number of inversions and translocations. The cause(s) of these rearrangements is (are) not known. A number of potentially powerful genetic tools designed to analyse the L. lactis genome have been constructed in recent years. These tools enable gene inactivation, gene replacement and gene recovery experiments to be readily carried out with this organism, and potentially with other lactic acid bacteria and Gram-positive bacteria. Integration vectors based on temperate phage attB sites and the random insertion of IS elements have also been developed for L. lactis and the intestinal lactobacilli. In addition, a L. lactis sex factor that mobilizes the chromosome in a manner reminiscent to that seen with Escherichia coli Hfr strains has been discovered and characterized. With the availability of this new technology, research into the genome of the lactic acid bacteria is poised to undertake a period of extremely rapid information accrual.     

Presence of mycobacteriophage I3-like DNA sequences in the genome of its host Mycobacterium smegmatis

The genomes of phage I3 and its host Mycobacterium smegmatis have been compared. From thermal melting studies the GC contents of DNA from mycobacteriophage I3 and its host M. smegmatis were found to be 66%. A new method, based only on the initial rates of reassociation, has been developed for calculating the DNA homology. Analysis of DNA reassociation kinetics suggested the presence of one equivalent of the phage I3 genome within the M. smegmatis genome. Southern analysis revealed the presence of almost all of the phage I3 specific sequences within the host genome.     

The chloroplast genome arrangement ofLobelia thuliniana (Lobeliaceae): Expansion of the inverted repeat in an ancestor of theCampanulales

A clone-bank ofSac I restriction fragments was constructed from the chloroplast DNA (cpDNA) ofLobelia thuliniana E. B. Knox (Lobeliaceae). These cloned fragments and a set of 106 clones spanning the tobacco chloroplast genome were used as probes to determine the cpDNA restriction fragment arrangement forSac I and six other restriction enzymes (BamH I,EcoR V,Hind III,Nci I,Pst I, andXho I) and the chloroplast genome arrangement ofL. thuliniana relative to tobacco, which has been fully sequenced and is collinear with the hypothesized ancestral genome arrangement of angiosperms. The results confirm and refine our previous understanding of the chloroplast genome arrangement in the large single-copy region (LSC) and reveal (1) a roughly 11 kilobase (kb) expansion of the inverted repeat (IR) into the small single-copy region (SSC) and (2) apparent sequence divergence of the DNA segment inL. thuliniana that corresponds to ORF1901 in tobacco. The expansion of the IR into the SSC is present in all other examined members ofLobeliaceae, Cyphiaceae, andCampanulaceae, which indicates that the IR expansion was an early event in the cpDNA evolution of theCampanulales. The IR expansion into the SSC was not present inSphenoclea, which additionally supports exclusion of this genus from theCampanulaceae.     

A Preliminary Linkage Map of the Tick, Ixodes scapularis

A linkage map of the Ixodes scapularis genome was constructed based upon segregation amongst 127 loci. These included 84 random amplified polymorphic DNA (RAPD) markers, 32 Sequence-Tagged RAPD (STAR) markers, 5 cDNAs, and 5 microsatellites in 232 F₁ intercross progeny from a single, field-collected P₁ female. A preliminary linkage map of 616 cM was generated across 14 linkage groups with one marker every 10.8 cM. Assuming a genome size of ∼10⁹ bp, the relationship of physical to genetic distance is ∼300 kb/cM in the I. scapularis genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence Heterogeneity of the Ten rRNA Operons in Clostridium perfringens

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA^Ile genes between the 16S and 23S rDNAs, and the other had a tRNA^Ile genes between the 16S and 23S rDNAs and a tRNA^Asn gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.     

Transposable elements as activators of cryptic genes in E. coli

The concept of transposable elements (TEs) as purely selfish elements is being challenged as we have begun to appreciate the extent to which TEs contribute to allelic diversity, genome building, etc. Despite these long-term evolutionary contributions, there are few examples of TEs that make a direct, positive contribution to adaptive fitness. In E.coli cryptic (silent) catabolic operons can be activated by small TEs called insertion sequences (IS elements). Not only do IS elements make a direct contribution to fitness by activating cryptic operons, they do so in a regulated manner, transposing at a higher rate in starving cells than in growing cells. In at least one case, IS elements activate an operon during starvation only if the substrate for that operon is present in the environment. It appears that E. coli has managed to take advantage of ISelements for its own benefit. This revised version was published online in July 2006 with corrections to the Cover Date.     

Restriction site map of the Chlorella virus PBCV-1 genome

The virus PBCV-1, which replicates in a Chlorella-like green alga, has a dsDNA genome. The DNA was mapped for BamHI, HindIII, and PstI restriction sites. The resulting map has a size of 333 kbp and is circular—indicating either covalently closed circular DNA or circularly permuted linear DNA. Several regions of repetitive DNA were also identified and located on the restriction map.     

The consensus land plant chloroplast gene order is present, with two alterations, in the moss Physcomitrella patens

Summary A restriction endonuclease cleavage site map for the enzymes ClaI and BglII, and a partial map for SacI, has been constructed for the chloroplast genome of the moss Physcomitrella patens (Hedw.) BSG. The plastid chromosome contains approximately 122 kb organized into small (21 kb) and large (82 kb) single-copy regions separated by two copies of a repeat sequence (9.4 kb) oriented in an inverted arrangement. Genes for 17 proteins and 2 ribosomal RNAs have been mapped using heterologous probes from corn, spinach, pea, and petunia. The general order and arrangement of the moss chloroplast genes are similar to the consensus land plant genome typified by that of spinach, with two major exceptions. First, there is an inversion of approximately 20 kb, bordered internally by psbA and atpH, and also containing the genes atpF and atpA. Second, rpl2 and rps19 have been relocated to a different position within the large single-copy region, adjacent to the 20 kb inversion.     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5-ATTTAAAT-3),PacI (5-TTAATTAA-3), andPmeI (5-GTTTAAAC-3) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996