Showing papers in "Molecular Genetics and Genomics in 1982"

Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis

Abstract: We have determined the nucleotide sequence of twoBacillus subtilis promoters (veg andtms) that are utilized by the principal form ofB subtilis RNA polymerase found in vegetative cells (σ55-RNA polymerase) and have compared our sequences to those of several previously reportedBacillus promoters Hexanucleotide sequences centered approximately 35 (the “-35” region) and 10 (the “-10” region) base pairs upstream from theveg andtms transcription startpoints (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed toEscherichia coli promoters Conformity to the preferred -35 and -10 sequences may not be sufficient to promote efficient utilization byB subtilis RNA polymerase, however, since three promoters (veg, tms andE coli tac) that conform to these sequences and that are utilized efficiently byE coli RNA polymerase were used with highly varied efficiencies byB subtilis RNA polymerase

pIJ101, a multi-copy broad host-range Streptomyces plasmid: Functional analysis and development of DNA cloning vectors

Abstract: Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit “lethal zygosis” and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.

Gene expression in Streptomyces : Construction and application of promoter-probe plasmid vectors in Streptomyces lividans

Abstract: Promoter-probe plasmid vectors were constructed for Streptomyces lividans using expression of the Escherichia coli chloramphenicol acetyltransferase gene as an indicator of promoter activity. These vectors have been used to isolate and to study the activity of DNA sequences that contain transcriptional control signals from Streptomyces, Bacillus licheniformis, E. coli, and Serratia marcescens. Studies of these promoter regions in heterospecific hosts indicate that genus or species-specific factors may present barriers to the expression of bacterial genetic material in certain heterologous cellular environments. While promoter regions isolated from E. coli, S. marcescens and B. licheniformis all appear to be recognized by the RNA polymerase of S. lividans, the Streptomyces transcriptional control signals isolated do not appear to function normally in E. coli.

Construction of a Schizosaccharomyces pombe gene bank in a yeast bacterial shuttle vector and its use to isolate genes by complementation

Abstract: A gene bank of partial Sau3A restriction fragments of S. pombe DNA has been constructed in the plasmid vector, pDB248', which is capable of high frequency transformation of S. pombe. Procedures are described which enable plasmids to be recovered from S. pombe by their reintroduction into E. coli. These methods have been used to detect the S. pombe genes lys 1+, ade 6+ and his 2+ in the gene bank by complementation of mutant gene functions, and to physically isolate the lys 1+ gene.

Plasmid determined nodulation and nitrogen fixation abilities in rhizobium phaseoli

Abstract: InRhizobium phaseoli strain 8002, the 190 Md plasmid pRP2JI which determines the ability to produce nitrogen-fixing nodules onPhaseolus beans (Nod+ Fix+) and the production of melanin on L-tyrosine-containing media (Mel+), was shown to be transmissible by conjugation to otherRhizobium strains. When pRP2JI was transferred to Nod- strains ofR. leguminosarum (which normally nodulates peas) the transconjugants gained the ability to nodulatePhaseolus beans and to make melanin. Out of 187 derivatives of strain 8002 carrying pRP2JI plasmids into which the transposon Tn5 had been inserted, six were Fix- Nod+ Mel+, one was Fix- Nod+ Mel- and four were Fix+ Nod+ Mel-. Three other derivatives of strain 8002 were Nod- Mel-; these had suffered deletions of c 30 Md in pRP2JI. Thus the genes for melanin production and nodulation appear to be closely linked, but melanin production is not necessary for the induction of nitrogen-fixing nodules onPhaseolus beans.

DNA from Agrobacterium-Rhizogenes Is Transferred to and Expressed in Axenic Hairy Root Plant-Tissues

Abstract: Axenic root tissue cultures were established from primary hairy roots induced on carrot and potato by Agrobacterium rhizogenes strain 15834. cDNA made towards poly-A+ RNA isolated from these tissues, hybridized with a limited number of well-defined fragments of the plasmid DNA present in the inciting A. rhizogenes strain. These data therefore demonstrate that at least part of the rootinducing (Ri) plasmid of Agrobacterium rhizogenes is transferred, stably maintained and expressed in hairy-root plant tissues and confirm that hairy roots are a special type of crown gall. The T-DNA in hairy-root cells appears to have several regions which are related in terms of sequence homology and probably also function to the T-DNA in octopine and nopaline crown gall tumours.

Repressor properties of the nifL gene product in Klebsiella pneumoniae

Abstract: Certain mutations in the nifL gene of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster resulted in altered nif regulaiton such that nitrogenase synthesis was no longer repressed by low levels of exogenous fixed nitrogen, by oxygen or by high temperature. Introduction of a plasmid with a nifL+ allele restored fixed nitrogen and oxygen repression. We therefore conclude that the nifL product acts as a nif-specific repressor in response to these effectors.

Cloning and Expression of a Bacillus-coagulans Amylase Gene in Escherichia-coli

Abstract: A partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage lambda host-vector system was shown to direct the synthesis of a thermostable alpha-amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000. The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused beta-lactamase-alpha-amylase protein with amylase activity.

Cytoplasmic hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions.

Abstract: Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.

Translation rates and misreading characteristics of rpsD mutants in Escherichia coli.

Abstract: Three ribosomal ambiguity (Ram) mutants, changed in ribosomal protein S4, have been examined with respect to elongation rate and misreading of translation in vivo and in vitro. Ram mutants increase misreading of nonsense codons in vivo, compared to wild type, between 2–50 times depending on the nature of the nonsense codon, its position, and which rpsD allele is present. Ram ribosomes also show an increased error frequency in vitro. The elongation rate of translation does not seem to be significantly changed, neither in vivo nor in vitro, irrespective of which rpsD allele is present. We suggest that there exists no general relationship between the accuracy and the overall speed of translation in Ram strains.

Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli

Abstract: Cellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42 degrees C but not at 30 degrees C. We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures. SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA+ B/r-derived recipient. SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA+. Each of the SC strains expresses SOS functions in a distinctively anomalous way. We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus. We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.

Promoters of the atp operon coding for the membrane-bound ATP synthase of Escherichia coli mapped by Tn10 insertion mutations.

Abstract: Transposon Tn10 insertion mutations in the oriC (replication origin) — atp (operon for the subunits of the membrane-bound ATP synthase) region of the Escherichia coli K12 chromosome were used to define promoters of the atp operon. The Tn10 insertions were first isolated and physically mapped on the specialized transducing phage λasn20 with a genome size of 23.5 MD, the insertion into which of the 6 MD Tn10 did not reduce packaging or stability. After transfer by recombination into the chromosome, a class of six Tn10 insertions was found to reduce growth yield and expression of the atp operon determined by quantitizing synthesis of the c-subunit of the ATP synthase. These six Tn10 insertions mapped within a 400 basepair segment of chromosomal DNA in front of the atpB gene. This segment contains the coding sequence for a 14KD polypeptide designated atpI. Complementation tests showed that the mutations are cis dominant and revealed that the 14 KD atpI gene product is not essential for biosynthesis and activity of the membrane bound ATP synthase. The insertions appear to block transcription into the atp genes located downstream. The insertions had different effects on atp operon expression: the ones closest to the atpB gene gave a 80%–90% decrease in c-subunit synthesis while those 150–200 basepairs further upstream only gave a 60%–70% decrease. These differential effects are taken to indicate the presence of three transcription start sites for the atp operon, a major promoter, designated atpIp, in front of the atpI gene and two minor ones, atpB1p and atpB2p, within the coding sequence atpI in front of the atpB gene. The allocation of atpIp coincides with potential transcription start signals found by DNA sequence analysis.

Conjugal Transfer of Nopaline and Agropine Ti-Plasmids- The Role of Agrocinopines

Abstract: The conjugative behaviour of nopaline and agropine Ti-plasmids has been investigated. Using a technique which avoids enrichment of transconjugants on a mating medium we have shown that preculture in the presence of agrocinopines A or B of donor strains harbouring nopaline Ti plasmids promotes plasmid transfer whereas preculture of the same strains in the presence of nopaline has no such effect. Similarly, preculture in the presence of agrocinopines C or D promotes Ti-plasmid transfer from strains harbouring agropine Ti-plasmids.

Lysis of Escherichia coli by induction of cloned ϕX174 genes

Abstract: The largest of the fragments produced by AluI digestion of ϕX174 RFI DNA comprises genes E and J as well as parts of genes D and F. This DNA fragment (1007 bp) was cloned into the lac z′ gene of plasmid pUR222. In the recombinant plasmid pUH12, transcription of the ϕX174 genes is controlled by the lac p-o region. Induction of the cloned genes by addition of the lac inducer, IPTG, resulted in lysis of the bacteria. Cloning of the corresponding AluI-fragment from ϕX174am3 DNA, carrying an amber mutation in gene E, showed that the expression of this gene alone is sufficient to trigger cell lysis. The time interval between the addition of IPTG and the onset of lysis depended on the concentration of the inducer, however, the rate of lysis was similar at all IPTG concentrations used.

Effects of bacteriophage f1 gene III protein on the host cell membrane.

Abstract: Plasmids which encode bacteriophage f1 coat protein genes VIII and III are responsible for a number of unusual properties suggesting that they have a drastic effect on the bacterial outer membrane. Analysis of several such recombinant plasmids and selection of mutant plasmids unable to cause this effect established that the properties were caused by gene III protein or its amino-terminal fragment.

Mobilization of a Rhizobium meliloti megaplasmid carrying nodulation and nitrogen fixation genes into other rhizobia and Agrobacterium

Abstract: The indigenous megaplasmid pRme41b of Rhizobium meliloti 41 was made susceptible to mobilization with the P-1 type plasmid pJB3JI by inserting the mobilization (mob) region of RP4 into it. First the mob region together with a kanamycin resistance marker was inserted in vitro into a fragment of pRme41b cloned into pBR322. The recombinant plasmids so formed (pAK11 and pAK12) were then mobilized into R. meliloti. Since these recombinant plasmids were unable to replicate in R. meliloti, selection for kanamycin resistant derivatives allowed the isolation of pRme41b::pAK11 or pRme41b::pAK12 cointegrates. It was shown that in the majority of these recombinants, pAK11 or pAK12 was integrated into the homologous fragment of pRme41b. The pRme41b cointegrates were transferred into nod-nif deletion mutants of R. meliloti 41 where it was shown that both Nod+ and Fix+ phenotypes could be restored. The pRme41b cointegrates were also transferred into two other Rhizobium strains and into Agrobacterium tumefaciens. The Rhizobium strains and A. tumefaciens carrying pRme41b formed nodules of variable size on Medicago sativa roots, indicating that at least the early steps of nodulation of M. sativa are coded by pRme41b and are expressed in these bacteria.

Effects of dosage alterations at the per locus on the period of the circadian clock of Drosophila

Abstract: The normal 24-h period of the circadian rhythms of locomotor activity and eclosion of Drosophila melanogaster is altered by changes in per gene dosage. Females with only one dose of per+ or pers (the 19-h short-period mutant allele) or per1 (the 29-h long-period mutant allele) have periods which are about 1–2 h longer than the corresponding females with 2 doses. Females with 3 doses of per+ and males with 2 doses of per+ or pers have periods which are 1/2 to 1 h shorter than the corresponding individuals without the extra dose. Males with three per+ doses have periods which are about 1.5 h shorter than wild-type males; additional per+ doses do not shorten period further. The observation that decreased per dosage lengthens period while increased dosage shortens period suggests that the long- and short-period mutations alter period by respectively decreasing and increasing per gene or gene product activity. The per+ dosage results and the complementation behavior of pers indicate that the hypermorphic phenotype of pers results from increased activity of the pers gene product rather than an overproduction of per+ product. This is the first report of such a mutant action in Drosophila.

Quantitative evaluation of recA gene expression in Escherichia coli.

Abstract: A recA::lac operon fusion was constructed using the phage Mu d(Ap, lac) in Escherichia coli to obtain precise measurements of the level of recA gene expression in various genetic backgrounds. The RecA protein normally represents 0.02% of total protein. This value is known to increase dramatically after treatments interrupting DNA synthesis; kinetic experiments showed that the rate of recA expression increases 17-fold within 10 min after UV irradiation or thymine starvation. In mutants affected in SOS regulation or repair the following observations were made: (i) the tif-1 mutation in the recA gene does not alter the basal level of recA expression, suggesting that it improves the protease activity of RecA; (ii) the lexA3 mutation does not create a “super-repressor” of recA; (iii) the tsl-1 mutation in the lexA gene makes the LexA protein a poor repressor of recA at 30°C (2.5-fold derepression) and a poor substrate for RecA protease (3-fold stimulation of recA expression by UV); (iv) the spr-55 amber mutation in the lexA gene causes a 30-fold increase in recA expression, higher than all inducing treatments, and this level cannot be further increased by nalidixic acid; (v) the zab-53 mutation at the recA locus, known to abolish tsl-mediated induction of recA expression, is trans-recessive and thus probably affects a regulatory site on the DNA; (vi) uvrA, B and C, recB and recF mutations do not increase the basal level of recA expression, suggesting that there are not sufficient spontaneous lesions to cause induction even when any one of these three repair pathways is inoperative.

Induction of E. coli recA protein via recBC and alternate pathways: Quantitation by enzyme-linked immunosorbent assay (ELISA)

Abstract: An enzyme-linked immunosorbent assay (ELISA) has been adapted to measure E. coli recA protein in the 1 to 10 ng range in whole-cell sonicates, membrane extracts, and osmotic shock fluid from 2x108 cells. The specific activity of recA protein is maintained at a relatively constant “basal” level (800 to 1,200 molecules per cell for wild-type E. coli in L-broth, salt-depleted broth and minimal media) during early-log and mid-log phase growth, but it increases by two- to ten-fold as the culture approaches saturation density. Nalidixate-induced levels are 20- to 50-fold higher, and 100-fold higher in a constitutive tif - spr -mutant. Induction of recA protein synthesis by nalidixic acid, which normally requires functional recBC enzyme, also occurs in recB -and recC -cells by pathways activated by mutation in the sbcA and sbcB indirect suppressors. In recB - sbcA -mutants, exonuclease VIII, the recE gene product, is required for induction of recA protein. Abolition of exonuclease I activity by mutation in sbcB allows induction of recA protein by nalidixate in recB -and recC -cells. Mutation in recF does not affect induction by nalidixate in RecBC+ cells, but it enables induction to occur in RecBC- cells, suggesting that recF gene product is involved in regulation of recA protein.

Molecular relationships among plasmids of Bacillus thuringiensis: conserved sequences through 11 crystalliferous strains.

Abstract: Screening for the plasmid content of 11 strains belonging to nine different serotypes ofB. thuringiensis was carried out by electron microscope examination and electrophoresis in agarose gels. All the strains contained at least two covalently closed, circular (CCC) DNA species. In one strain (berliner 1715), 17 extrachromosomal elements could be distinguished with regard to their size, ranging from 3.9 to 180 Mdal. Southern hybridisation experiments showed that most of these plasmids fell into two categories (inferior to 15 Mdal and superior to 15 Mdal) which have no homology between them. Within these two size groups there is partial conservation of DNA sequences through various serotypes. Further relationships among the plasmids were investigated by a two dimensional version of the Southern's blotting technique.

Cytoplast-protoplast fusion for interspecific chloroplast transfer in Nicotiana

Abstract: Protoplasts of Nicotiana tabacum (SR1), carrying a maternally-inherited streptomycin resistance mutation, were enucleated by centrifugation through a Percoll gradient. The resulting cytoplasts containing resistant plastids, were fused with sensitive Nicotiana plumbaginifolia protoplasts. The SR1 cytoplasts, having no nuclei, were unable to form calli. All resistant clones recovered after fusion-induction were therefore supposed to be derived from interspecific cytoplast-protoplast fusion. N. plumbaginifolia plants regenerated in 17 out of the 75 resistant clones studied. Plants obtained from eight of these clones were resistant to streptomycin and inherited the resistance maternally, as expected when transferring SR1 plastids into the N. plumbaginifolia nuclear background. Plastid transfer in these plants has been confirmed by the EcoRI restriction pattern of the chloroplast DNA. In nine clones N. plumbaginifolia plants were sensitive although obtained from initially resistant clones. This phenomenon is explained by the maintenance of plastid heterogeneity on the selective streptomycin medium, and formation of plants from sensitive sectors on the non-selective regeneration medium. SR1 protoplasts, originally present as “contaminants” in the cytoplast preparation (2–7%) did not form colonies (or very rarely) after polyethylene glycol treatment. The nuclei from such protoplasts were recovered, however, in the interspecific somatic hybrids (56 clones), and in segregants having the SR1 nucleus but some cytoplasm from N. plumbaginifolia (2 clones). The majority (about 80%) of the recovered resistant clones therefore acquired the streptomycin resistance factor from the rare (2–7%) “contaminating” SR1 protoplasts. This is explained by the protoplasts being more stable during fusion induction.

Nitrate reductase deficient cell lines from haploid protoplast cultures ofNicotiana plumbaginifolia

Abstract: Nitrate reductase deficient (NR-) cell lines were selected indirectly by their resistance to 40 mM chlorate in protoplast cultures of haploidNicotiana plumbaginifolia. Frequency of the chlorate resistant clones was 5.8×10-5 in non-mutagenized cultures, which could be increased up to 25 times by treatment with N-ethyl-N-nitrosourea (NEU) or gamma irradiation.

Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein.

Abstract: We have used a sensitive gel electrophoresis assay to detect the products of Escherichia coli RecA protein catalysed strand exchange reactions between gapped and duplex DNA molecules. We identify structures that correspond to joint molecules formed by homologous pairing, and show that joint molecules are converted by RecA protein into heteroduplex monomers by reciprocal strand exchanges. However, strand exchanges only occur when there is a 3'-terminus complementary to the single stranded DNA in the gap. In the absence of a complementary free end, the two DNA molecules pair and short heteroduplex regions are formed by localised interwinding.

Nicotiana chloroplast genome

Abstract: N. accuminata has a chloroplast genome of 171 Kb which is larger than 160 Kb reported for N. tabacum. A physical map of the former has been constructed by SalI, BglI, and PvuII enzymes in comparison with N. tabacum. They both share identical restriction map except in the extra segment of N. accuminata. This extra segment is located on the right-hand border of an inverted repeat in the large-single copy region of N. accuminata. It contains the following restriction sites: two for BglI and SmaI; three for SalI and XhoI. The size of inverted repeat measured by electron microscope is 22.67±0.78 Kb and 19.28±0.61 Kb for N. accuminata and N. tabacum, respectively. This is the first case where a larger inverted repeat region accompanied by a larger genome size was found among over 30 Nicotiana species studied thus far.

Damage to DNA induces expression of the ruv gene of Escherichia coli.

Abstract: Regulation of the ruv gene of E. coli was studied using phage Mud (Ap lac) to obtain a fusion of the lac genes to the ruv promoter. Beta-galactosidase synthesis in the ruv-lac fusion strain was induced by mitomycin C and other agents that damage DNA. The induction of beta-galactosidase could be altered by mutations either in lexA or recA from which it is concluded that ruv is regulated by lexA repressor. A possible function of ruv in promoting cell recovery following damage to DNA is discussed.

Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids

Abstract: Growth in a chemostat of the 3-chlorobenzoatepositive Pseudomonas putida cells harboring the plasmid pAC25, in presence of cells harboring the TOL plasmid, allows emergence of cells that can also utilize 4-chlorobenzoate (4Cba). Isolation of plasmid DNA from such cells demonstrate the deletion of a 11kb (Kilobase pair) EcoR1 fragment from the pAC25 plasmid; a portion of the TOL plasmid (41.5 kb TOL*) is also found to be transposed onto the chromosome of such cells. Further enrichment of the 4-chlorobenzoate-positive cells with 3,5-dichlorobenzoate (3,5-Dcb) as a sole carbon source has produced cells that can also slowly utilize 3,5-dichlorobenzoate. Isolation of plasmid DNA from such cells demonstrates the appearance of a second plasmid (pAC29). Restriction hybridization of pAC29 EcoRI fragments with pAC25 and TOL demonstrates that pAC29 is derived primarily by duplication of a segment of the pAC27 plasmid and a fragment from TOL, with further mutational divergence. Southern hybridization of the EcoRI-digested chromosomal DNA with 32P-labeled TOL, pTS11 and pTS71 plasmid DNAs demonstrates the presence of the TOL* transposon containing xylD, G, E and F genes in both 4Cba+ (pAC27+) and 3,5-DCb+ (pAC27+, pAC29+) cells. Isolation of plasmid DNA from 3,5-Dcb+ faster growing variants, obtained from slow-growing pAC27+ pAC29+ cells, demonstrates the presence of a single type of plasmid, with identical size and EcoRI digestion profile as pAC27. The implications of gene duplications and subsequent homologous recombination with regard to the biochemical pathway of 3,5-dichlorobenzoate degradation have been discussed.

Recombination between short direct repeats in a recA host.

Abstract: Spontaneous deletion derivitives of plasmid pHV14, a recombinant of pBR322, have been isolated in the recA strain HB101. About 2.5 kilobases (Kb) of DNA has been lost in each of the four plasmids described including, in two cases the region involved in control of copy number and the initiation of replication.

The sites of action of the two copy number control functions of plasmid R1

Abstract: Two negatively acting functions - the CopA-RNA and the CopB protein - are involved in the control of replication of plasmid R1 They both act as inhibitors of expression of a gene, repA, which seems to be positively required for autonomous plasmid replication Here we show that the two control functions act separately and independently The CopB protein represses initiation of transcription of the repA gene, and its target site lies within a 60 base pair region containing the repA promoter The CopA-RNA acts downstream of the repA promoter in the leader sequence containing the copA gene itself, preceding the repA structural gene Measurements of RepA-beta-galactosidase expression from wild-type and a copA mutant fusion hybrid in the presence of extra copies of the respective copA genes show that a point mutation affecting the activity of the CopA-RNA can also affect CopA target properties It is therefore concluded that the target site for the CopA-RNA resides within the copA gene in a small region encoding the loop of a stem-loop structure in the CopA-RNA In addition, the data indicate a direct nucleic acid-nucleic acid interaction as the basis for the CopA inhibitor activity

The organization and regulation of the pyrBI operon in E. coli includes a rho-independent attenuator sequence.

Abstract: In this paper we describe the structural organization of pyrBI, and provide a detailed analysis of its regulatory region including its DNA sequence.

Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids

Abstract: The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm-3 and size of approximately 156 kbp are similar to those of ptDNA from other higher plants. The Sal I restriction patterns were indistinguishable; however, those obtained with Kpn I, Pst I, and Eco RI disclosed that each species possesses a unique ptDNA. These observations suggest a relatively recent divergence of both species. Of the twelve hybrid lines screened, eight contained exclusively potato ptDNa and four contained only tomato ptDNA at a 0.1–3% level of detection. Rearrangements of modifications of DNA were not detected. The plastid identities of three hybrid lines that had previously been analyzed by isoelectric focusing of RuBPcase subunits (Melchers et al. 1978) agreed with those determined by restriction endonuclease analysis.