Analysis of the plastid DNA in an Oenothera plastome mutant deficient in ribulose bisphosphate carboxylase

Summary Plastid DNA of the light green Oenothera plastome mutant sigma, from plastome I, which is deficient in ribulose bisphosphate carboxylase, has been compared with wild-type chloroplast DNA from plastome I and the related plastome IV. For this, double digestions with the restriction endonucleases Sal I, Pst I and Kpn I were used. Chloroplast DNA from plastomes I and IV differs in the sizes of several fragments, with the changes being from under 0.1 to about 0.6 Md in size. In the cleavage patterns of the mutant DNA compared to the wild-type DNA from plastome I, the only differences observed are two possible deletions of less than 0.1 Md from a fragment known to partly cover the genes for the ribosomal RNAs and from a fragment located in the small single-copy region of the molecule. It is concluded that the ribulose bisphosphate carboxylase deficiency in this mutant is not caused by a major deletion in the plastid DNA.     

MEI1, an Arabidopsis gene required for male meiosis: isolation and characterization

 The T-DNA tagged mutant gene of Arabidopsis thaliana, mei1, produces after meiosis an abnormal tetrad, consisting of five to eight microspores of varying sizes and DNA contents. Plant DNA flanking the inserted T-DNA was isolated by inverse PCR. An approximately 16-kb DNA fragment spanning the T-DNA insertion site was isolated by screening a wild-type genomic library, using the plant flanking DNA as a probe. Using RT-PCR and RNA isolated from very young flower buds, a cDNA fragment was obtained. Nucleotide sequence comparison of the cDNA and the genomic sequence in this region indicated a gene which contained two introns. The 5′ and 3′ splice sites of neither intron comply with the :GU...AG: rule. In the mutant, the T-DNA had inserted into one of the introns. The deduced sequence of the MEI1 wild-type gene, which contains 89 amino acids, shows possible similarity with the human acrosin-trypsin inhibitor, HUSI-II, and is about the same size. Two wild-type DNA fragments, both extending over the T-DNA insertion site, were introduced into mutant plants by Agrobacterium-mediated transformation and plants were selected for both hygromycin and kanamycin resistance. Several independent male-fertile transformants were obtained with one of the DNA fragments. The fragment showing complementation of the mutant phenotype indicated that the sequence with similarity to the acrosin-trypsin inhibitor is MEI1. Within the 16-kb genomic fragment two other genes were identified; one showed no overall similarity to any protein sequence in the database and the other had almost complete identity with an Arabidopsis-transcribed sequence tag with similarity to ACC oxidase. Double mutants between mei1 and qrt1 were made, permitting better characterization of the mei1 phenotype because the individual microspores continued to be held together after callose dissolution. Received: 21 April 1998 / Revision accepted: 11 June 1998     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.     

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G+C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome. Correspondence to: C. Diviès     

Locations and stability of Agrobacterium-mediated T-DNA insertions in the Lycopersicon genome

Summary The genomic distribution and genetic behavior of DNA sequences introduced into the tomato genome by Agrobacterium tumefaciens were investigated in the backcross progeny of 10 transformed Lycopersicon esculentum x L. pennellii hybrids. All transformants were found to represent single locus insertions based on the co-segregation of restriction fragments corresponding to the T-DNA left and right border sequences in the backcross progeny. Isozyme and restriction fragment length polymorphism (RFLP) markers were used to test linkage relationships of the insertion in each backcross family. The T-DNA inserts in 9 of the 10 transformants were mapped in relation to one or more of these markers, and each mapped to a different chromosomal location. Because only one insertion did not show linkage with the markers employed, it must be located somewhere other than the genomic regions covered by the markers assayed. We conclude that Agrobacterium-mediated insertion in the Lycopersicon genome appears to be random at the chromosomal level. No discrepancies were found between the T-DNA genotype and the nopaline phenotype in the 322 backcross progeny of the nopaline positive transformants. Backcross progeny of two nopaline negative transformants showed incomplete correspondence between the T-DNA genotype and the kanamycin resistance phenotype. No alteration of T-DNA was observed in progeny showing a discrepancy between T-DNA and kanamycin resistance. However, two kanamycin resistant progeny plants of one of these two transformants possessed altered T-DNA restriction patterns, indicating genetic instability of the T-DNA in this transformant.Journal article no. 1223 of the New Mexico Agricultural Experiment Station     

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     

Optimization of the genotyping‐by‐sequencing strategy for population genomic analysis in conifers

Flexibility and low cost make genotyping‐by‐sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI‐MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference‐free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000–11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking.     

Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes

A restriction map of the 2.8-Mb genome of the unicellular eukaryote Encephalitozoon cuniculi (phylum Microspora), a mammal-infecting intracellular parasite, has been constructed using two restriction enzymes with 6 bp recognition sites (BssHII and MluI). The fragments resulting from either single digestions of the whole molecular karyotype or double digestions of 11 individual chromosomes have been separated by two-dimensional pulsed field gel electrophoresis (2D-PFGE) procedures. The average distance between successive restriction sites is ~19 kb. The terminal regions of the chromosomes show a common pattern covering ~15 kb and including one 16S–23S rDNA unit. Results of hybridisation and molecular combing experiments indicate a palindromic-like orientation of the two subtelomeric rDNA copies on each chromosome. We have also located 67 DNA markers (clones from a partial E.cuniculi genomic library) by hybridisation to restriction fragments. Partial or complete sequencing has revealed homologies with known protein-coding genes for 32 of these clones. Evidence for two homologous chromosomes III, with a size difference (3 kb) related to a subtelomeric deletion/insertion event, argues for diploidy of E.cuniculi. The physical map should be useful for both the whole genome sequencing project and studies on genome plasticity of this widespread parasite.     

Number and Accuracy of T-DNA Insertions in Transgenic Banana (Musa spp.) Plants Characterized by an Improved Anchored PCR Technique

Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique. The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments. Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant, indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied.     

Optimization of transverse alternating field electrophoresis for strain identification of Leuconostoc oenos

Genomic DNA of several strains oof oenological lactic bacteria belonging to the species Lactobacillus plantarum, Leuconostoc oenos and Pediococcus pentosaceus was digested by the rare-cutting endonucleases ApaI and SmaI. The restriction products were separated by transverse alternating field electrophoresis (TAFE). The size of the genome of L. oenos estimated by adding the molecular size of the ApaI fragments was on average 1320 kb. A strong polymorphism was observed between the strains, which could be easily differentiated except for two industrial strains of L. oenos. A simple modification of the TAFE apparatus is proposed to improve the separation of the DNA fragments. Correspondence to: J.-N. Hallet     

Sequence comparison of two codominant RAPD markers in Brassica nigra: deletions,substitutions and microsatellites

Summary Two RAPD fragments segregating codominantly were investigated in a F₂ population of Brassica nigra. Southern hybridization of these DNA fragments to genomic B. nigra DNA digested with several endonucleases revealed similar restriction profiles. Sequencing of the two fragments disclosed 93% homology. The differences were due mainly to an internal 41 nucleotide deletion in one of the fragments. Minor deletions of one to three bases, including a microsatellite of CTT motif were also observed. In addition, base substitutions, mostly transitions were detected. These relatively small differences suggested that the two RAPD products were indeed different versions of the same sequence. The larger fragment of 1154 bp was denominated A05¹ whereas the shorter one, denominated A05², had 1116 bp.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

A mutant rat major histocompatibility haplotype showing a large deletion of class I sequences

The LEW.1LM1 inbred rat strain, which has been derived from a (LEW×LEW.1W) F₂ hybrid, carries a major histocompatibility (RT1) haplotype which is distinct from that of the LEW strain (RT1 ¹) in that certainRT1.C region-determined class I antigens are not expressed. Here we show that this phenotypic defect is due to genomic deletion of about 100 kb of theRT1.C region. Certain deleted DNA fragments have been cloned from the wild-type DNA into the EMBL4 vector. Five clones have been characterized and are shown to possess different restriction maps and to each carry a single stretch of class I cross-hybridizing sequences. Probes derived from the non-class I coding part of two clones detect fragments which are present in the wild-type but absent from thelm1 mutant. The type of deletion described here in the rat is discussed in the context ofH-2D/Q deletions in the mouse.     

Segments of mitochondrial DNA of yeast carrying antibiotic resistances

Summary Mitochondrial DNA was isolated from an oligomycin-resistant petite mutant of yeast, Saccharomyces cerevisiae. It had repeated sequences of 3600 base pairs. This segment was about one twentieth of the whole mtDNA of wild type yeast, which had a size of 74 kilo base pairs.This segment of mtDNA had one cleavage site for a restriction endonuclease, Hind II, which was more resistant to cleavage than the other Hind II sites in wild type mtDNA. It had two cleavage sites for Hha I and gave two Hha fragments, which were arranged alternatively. Digestion with Hae III gave four fragments and these fragments were mapped.Mitochondrial DNA of this mutant showed a loss of heterogeneity in a melting profile. It melted within a narrow range of temperature, which was similar to that of poly dA·poly dT. Its differential melting curve was significantly different from that of wild type mtDNA.Mapping of mtDNA of a wild type yeast was carried out with restriction endonucleases. Fragments of mtDNA, which were isolated from petites carrying oligomycin-erythromycin-chloramphenicol-resistance and erythromycin-chloramphenicol resistance were also mapped. Loci of oligomycin-resistance, erythromycin-resistance and chloramphenicol-resistance were investigated based on the maps of Eco R I fragments and Hind II fragments.     

The plastome of a brown alga,Dictyota dichotoma

Summary Plastids of the brown algaDictyota dichotoma contain a single homogeneous DNA species which bands at a buoyant density of 1.693 g/cm³ in neutral CsCl equilibrium density gradients. The corresponding nuclear DNA has a density of 1.715 g/cm³. The molecular size of the plastid DNA is 123 kbp as calculated by both electron microscopy of spread intact circular molecules and gel electrophoresis following single and double digestions with various restriction enzymes. A restriction map has been constructed using the endonucleases Sal I, Bam HI, and Bgl II which cleave theDictyota plastome into 6, 12, and 17 fragments, respectively. No large repeated regions, as found in chlorophycean andEuglena plastid DNAs, were detected.Dictyota dichotoma is the first member from the chlorophyll c-line of the algal pedigree for which a physical map of plastid DNA has been established. Dedicated to Professor Dr. W. Stubbe on the occasion of his 65th birthday.     

Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes.

A macrorestriction map representing the complete physical map of the Rhodobacter sphaeroides 2.4.1 chromosomes has been constructed by ordering the chromosomal DNA fragments from total genomic DNA digested with the restriction endonucleases AseI, SpeI, DraI, and SnaBI. Junction fragments and multiple restriction endonuclease digestions of the chromosomal DNAs derived from wild-type and various mutant strains, in conjunction with Southern hybridization analysis, have been used to order all of the chromosomal DNA fragments. Our results indicate that R. sphaeroides 2.4.1 carries two different circular chromosomes of 3,046 +/- 95 and 914 +/- 17 kilobases (kb). Both chromosome I (3,046 kb) and chromosome II (914 kb) contain rRNA cistrons. It appears that only a single copy of the rRNA genes is contained on chromosome I (rrnA) and that two copies are present on chromosome II (rrnB, rrnC). Additionally, genes for glyceraldehyde 3-phosphate dehydrogenase (gapB) and delta-aminolevulinic acid synthase (hemT) are found on chromosome II. In each instance, there appears to be a second copy of each of these genes on chromosome I, but the extent of the DNA homology is very low. Genes giving rise to enzymes involved in CO2 fixation and linked to the gene encoding the form I enzyme (i.e., the form I region) are on chromosome I, whereas those genes representing the form II region are on chromosome II. The complete physical and partial genetic maps for each chromosome are presented.     

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.     

Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942

Summary In a temperature-sensitive, high CO₂-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41° C). In contrast, inorganic carbon uptake and ribulose-1,5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb Pstl fragments. These 4 fragments overlapped only in a 0.4 kb HindIII-PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis.     

Physical mapping of plasmid pDB101: A potential vector plasmid for molecular cloning in streptococci

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site. Plasmid pDB101 meets all requirements essential for a potential molecular cloning vehicle in streptococci; i.e., single restriction sites, a MLS selection marker, and a multiple plasmid copy number. The vector plasmid described here makes it possible to clone selectively any fragment of DNA cleaved with EcoRI, HpaII, or KpnI, or since the sites are close to each other in map position, any combination of two of these restriction enzymes.