Molecular cloning of tobacco chloroplast ribosomal RNA genes

Summary Tobacco chloroplast ribosomal RNAs were shown to be hybridized with two EcoRI fragments of tobacco chloroplast DNA. These DNA fragments having molecular weights of 1.9x10⁶ and 2.8x10⁶ daltons were cloned using the bacterial plasmid pMB9 as a vector and E. coli HB101 as host bacteria. The recombinant plasmids containing either or both of these fragments were constructed and characterized.Abbreviations rRNA ribosomal RNA - EDTA ethylenediamine tetraacetic acid - SSC 0.15 M NaCl-0.015 M sodium citrate - EcoRI and HindIII restriction endonucleases isolated from E. coli RY13 and Haemophilus influenzae Rd, respectively     

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     

A complete cleavage map of Neurospora crassa mtDNA obtained with endonucleases Eco RI and Bam HI.

A physical map of Neurospora crassa mitochondrial DNA has been constructed using specific fragments obtained with restriction endonucleases. The DNA has 5 cleavage sites for endonuclease Bam HI, 12 for endonuclease Eco RI and more than 30 for endonuclease Hind III. The sequence of the Eco RI and Bam HI fragments has been established by analysis of partial fragments. By digestion of the Eco RI fragments with Bam HI, a complete overlapping map has been constructed. The position of the largest Hind III fragment on this map has also been determined. The map is circular and the added molecular weight of the fragments is 40 - 10(6), which is in good agreement with earlier measurements on intact DNA, using the electron microscope.     

Nucleotide polymorphism in chloroplast dna of Nicotiana debneyi

Summary EcoR1 restriction endonuclease analysis of chloroplast DNA isolated from several distinct populations of Nicotiana debneyi has revealed a naturally occurring polymorphism. The chloroplast DNA of seven of the nine populations analysed possessed an additional EcoRl site. The origin of the additional restriction endonuclease fragments was confirmed by hybridisation of [³² P]-cRNA to fractionated EcoRl restricted chloroplast-DNA fragments adsorbed to nitrocellulose filters. Reciprocal f₁ hybrids between plants carrying the variant chloroplast-DNA's confirmed maternal inheritance of chloroplast-DNA.Communicated by G. Melchers and D. von Wettstein     

Glutathione production by Escherichia coli cells with hybrid plasmid containing tandemly polymerized genes for glutathione synthetase

Summary A DNA fragment containing the structural and promoter regions of glutathione synthetase (GSH II) gene (gsh II) from Escherichia coli B were polymerized. The dimeric and trimeric DNA fragments obtained were inserted into Bam HI site of vector plasmid pBR325 and the resulting hybrid plasmids were designated pGS401-02 and pGS401-03, respectively. The GSH II activity of E. coli cells with these hybrid plasmids increased depending on the number of the genes (gsh II) contained. To construct hybrid plasmids useful for glutathione production, another DNA fragment with a gene (gsh I) for -glutamylcysteine synthetase (GSH I) from E. coli B was inserted into Pst I sites of pGS401-02 and pGS401-03 and the hybrid plasmids obtained (pGS501-12 and pGS501-13, respectively) were introduced into E. coli B cells. Although the glutathione-producing activities of the cells with these plasmids were little improved as compared with that of cells with the hybrid plasmid (pGS501-11) containing both gsh I and gsh II because of the low activity of GSH I, our method has brought to light a new type of gene amplification.     

Cloning of the protective antigen gene of Bacillus anthracis

The tripartite protein toxin of Bacillus anthracis consists of protective antigen (PA), edema factor (EF), and lethal factor (LF). As a first step in developing a more efficacious anthrax vaccine, recombinant plasmids containing the PA gene have been isolated. A library was constructed in the E. coli vector pBR322 from Bam HI-generated fragments of the anthrax plasmid, pBA1. Two clones producing PA were identified by screening lysates with ELISA (enzyme-linked immunosorbent assay). Western blots revealed a full-size PA protein in the recombinant E. coli, and a cell elongation assay demonstrated biological activity. Both positive clones had a 6 kb insert of DNA, which mapped in the Bam HI site of the vector. The two inserts are the same except that they lie in opposite orientations with respect to the vector. Thus PA is encoded by the plasmid pBA1.     

An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system

The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.     

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

Cloning and Expression of a Gene for an 87-kDa β-1,3-Glucanase of Bacillus circulans IAM1165 in Escherichia coli K-12

Partially Sau3AI-digested fragments of chromosomal DNA from Bacillus circulans IAM1165, a high producer of β-1,3-glucanases able to lyse fungal cell walls, were inserted into a BamHI site of the plasmid vector pHSG399. A gene for the glucanase was cloned in Escherichia coli K-12 by the shotgun method. An 8-kb inserted DNA directed synthesis of an 87-kDa endo-β-1,3-n-glucanase in E. coli. The β-glucanase gene was in a 2.6-kb EcoRI-SmaI segment within the insert DNA. The enzyme activity was found mainly in the periplasmic fraction of E. coli carrying the gene.     

Isolation of yeast tRNALeu genes. DNA sequence of a cloned tRNALeu3 gene.

A library of cloned yeast DNA fragments generated by digestion of yeast DNA with the restriction endonuclease Bam HI has been screened by colony hybridization to total yeast [32P]tRNA. Four hundred colonies carrying yeast tRNA genes were isolated. By hybridization to 125I-tRNALeu3, we have isolated from this collection 14 colonies carrying fragments containing yeast tRNALeu genes. The size of the yeast Bam HI inserts ranged from 2.45 x 10(6) to 14 x 10(6) daltons. One of these fragments was mapped in detail by restriction endonuclease digestion and hybridization to 125I-tRNALeu3. The presence of a tRNALeu3 gene was confirmed by DNA sequence. The results indicate that the tRNALeu3 coding region is not co-linear with the tRNALeu3. An intervening tract of 33 base pairs interrupts the coding sequences 1 base pair past the anticodon coding region. The putative structure of a tRNALeu3 precursor is deduced in which the anticodon base pairs with residues from the intervening sequence.     

Stability of cloned Brassica napus chloroplast DNA fragments in the cyanobacterium Anacystis nidulans R2

Summary Brassica napus (cv. Triton) chloroplast (cp) DNA BamHI gragments were inserted into a bacteria-cyanobacteria shuttle vector pCB4. The chloroplast genomic library was screened in Escherichia coli and 28 individual clones, which represent 94% of the total chloroplast genome, were isolated. Cyanobacterium Anacystis nidulans R2 was transformed with each member of the clone bank by selection for ampicillin resistance. A study of transformation efficiency showed dramatic variation (up to 200-fold) among recombinant clones. Furthermore, plasmid DNA reisolated from some cyanobacterial transformants exhibited instability. Variations in transformation efficiency and plasmid instability were shown to be DNA sequence specific. B. napus cpDNA clones were thus classified into three types according to their stability in the cyanobacterial host.     

Molecular cloning of restriction fragments and construction of a physical and genetic map of the Escherichia coli plasmid R538-1.

A genetic and physical map of Escherichia coli plasmid R538-1 was constructed using restriction endonucleases and molecular cloning techniques. R538-1 DNA was cleaved into 12 fragments by endonuclease · R · EcoRI, 6 fragments by endonuclease R · HindIII, and 3 fragments by endonuclease R · BamHI. The order of these fragments was determined by standard restriction fragment mapping techniques. Endo · R · EcoRI, endo · R · HindIII, endo · R · BamHI, and endo · R · PstI fragments obtained from R538-1 and ColE1-derived plasmids (pMB9, ColE1Ap^r, and pBR322) were ligated in vitro and used to transform E. coli C600. Transformants were selected for antibiotic resistance markers carried by R538-1. Analysis of the R538-1 fragments contained in these hybrid plasmids permitted the construction of a genetic map of the R538-1 plasmid. The genetic map of this plasmid is very similar to that of plasmid R100.     

Karyotypic and chloroplast genomic diversity inMedicago sect.Lupularia (Leguminosae)

Studies were made on the chromosome complements and chloroplast genomes ofMedicago lupulina andM. secundiflora, which comprise sectionLupularia ofMedicago. Both types of analyses indicated more substantial differences between these species than suggested by external morphology.Medicago lupulina has a relatively asymmetrical karyotype in terms of centromeric position and relative length. The karyotype ofM. secundiflora is comparatively more asymmetrical in centromeric position and reduced in absolute size but exhibits greater symmetry in relative length. The restriction endonuclease fragmentation patterns of the chloropiast DNA of these two species (with Bam HI, Eco RI, Bgl II, and Xho I) show little similarity, with only 17% of the fragments matching in size. The lack of interspecific congruence among data of morphology, karyology and cpDNA inLupularia is contrary to consistency exhibited among these data inMedicago subsect.Intertextae.     

Complete cloning of the chloroplast genome of safflower in λ EMBL3 and mapping of 23S and 16S rRNA genes

Summary A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of EMBL3. Seventeen recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.     

Additional Volatile Compounds Produced by Pyrolysis of Sulfur-containing Amino Acids

Bacillus circulans WL-12, a yeast and fungal cell wall lytic bacterium, secretes a variety of polysaccharide degrading enzymes into the culture medium. When β-1,3-glucanase was induced with pachyman, a β-1,3-glucose polymer obtained from the tree fungus Poria cocus Wolf, six distinct active molecules of the enzyme with different molecular weights were detected in the culture supernatant of this bacterium. Molecular cloning of one of the β,3-gIucanase genes into E. coli was achieved by transforming E. coli HB101 cells with recombinant plasmids composed of chromosomal DNA fragments prepared from B. circulans WL-12 and the plasmid vector pUC 19. A recombinant plasmid containing 4.4 kb of inserted DNA in the Pst I site of pUC 19, designated as pNT003, conferred the ability to degrade pachyman on E. coli cells. The presence of pNT003 was harmful for E. coli cells and caused cell lysis, especially at higher temperatures of cultivation. β,3-Glucanase activity detected in E. coli was mainly recovered in the periplasmic fraction when cell lysis did not occur. SDS-PAGE analysis revealed that the periplasmic fraction contained four active molecules of β-1,3-glucanase which corresponded to four of the six active molecules produced by B. circulans WL-12.     

Cloning and expression of the Bacillus subtilis phage SPP1 in E. coli. I. Construction and characterization of lambda/SPP1 hybrids

Summary We have constructed /SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.Part of this work was taken from the doctoral thesis of E.P.A. submitted to the Freie Universität, Berlin 1979     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis

Summary Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tc^r gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8–9 Kbp in the BamHI and 4–5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec⁺ recipients. Loss of sequences from hybrid plasmids was not prevented in a r ^- m ^- recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.     

Cloning of bacteriophage PM2 DNA in Escherichia coli K12.

Summary DNA fragments of phage PM2 restricted with Hin dIII endonuclease was cloned in the vector pBR 322 in an Escherichia coli K 12 host. The attempt to clone full length PM2 DNA restricted with PstI endonuclease has been unsuccesful. From six randomly chosen recombinant clones DNA was purified and analysed with EcoRI, PstI and HindIII endonucleases. The physical map of three chimeric plasmids was unequivocally established. It was shown, that the whole PM2 genome was cloned, although in separate fragments. However, most of the recombinant clones were instable in the absence of selective pressure.