Changes of ploidy during the Azotobacter vinelandii growth cycle.

The size of the Azotobacter vinelandii chromosome is approximately 4,700 kb, as calculated by pulsed-field electrophoretic separation of fragments digested with the rarely cutting endonucleases SpeI and SwaI. Surveys of DNA content per cell by flow cytometry indicated the existence of ploidy changes during the A. vinelandii growth cycle in rich medium. Early-exponential-phase cells have a ploidy level similar to that of Escherichia coli or Salmonella typhimurium (probably ca. four chromosomes per cell), but a continuous increase of DNA content per cell is observed during growth. Late-exponential-phase cells may contain > 40 chromosomes per cell, while cells in the early stationary stage may contain > 80 chromosomes per cell. In late-stationary-phase cultures, the DNA content per cell is even higher, probably over 100 chromosome equivalents per cell. A dramatic change is observed in old stationary-phase cultures, when the population of highly polyploid bacteria segregates cells with low ploidy. The DNA content of the latter cells resembles that of cysts, suggesting that the process may reflect the onset of cyst differentiation. Cells with low ploidy are also formed when old stationary-phase cultures are diluted into fresh medium. Addition of rifampin to exponential-phase cultures causes a rapid increase in DNA content, indicating that A. vinelandii initiates multiple rounds of chromosome replication per cell division. Growth in minimal medium does not result in the spectacular changes of ploidy observed during rapid growth; this observation suggests that the polyploidy of A. vinelandii may not exist outside the laboratory.     

Characterization of Azotobacter vinelandii deoxyribonucleic acid and folded chromosomes.

The properties of Azotobacter vinelandii deoxyribonucleic acid (DNA) and folded chromosomes were studied and compared to those of Escherichia coli as a standard. Based on melting temperature and buoyant density measurements, the guanosine + cytosine content of purified A. vinelandii DNA was 65%, whereas that of E. coli DNA was 50%. The results of renaturation studies showed that the unique DNA sequence lengths of the two organisms were similar with Cot1/2 values of 7.3 +/- 0.4 mol.s/liter and 7.5 +/- 0.3 mol.s/liter, respectively, for A. vinelandii and E. coli. Folded chromosomes of A. vinelandii sedimented in a centrifugal field at a rate identical to those derived from E. coli, 1,600 to 1,700S. Based on the DNA content per cell and the mass of a single genome, A. vinelandii contains at least 40 chromosomes per cell.     

TheAzotobacter vinelandii chromosome

The chromosome ofAzotobacter vinelandii was digested with the restriction endonucleasesSpeI (5’-ACTAGT),DraI (5’-TTTAAA) andAsel (5’-ATTAAT) and the products were separated by pulsed-field gel electrophoresis. The sum of the sizes of the restriction fragments comes to around 4.5 megabasepairs. Our earlier studies had revealed the presence of about 80 copies ofnifH, nifD, nifK andleuB genes in a log-phase cell ofA. vinelandii. To determine whether there are multiple identical chromosomes inA. vinelandii or one large chromosome with identical segments joined in tandem, we have subjected gamma-irradiated DNA ofA. vinelandii andEscherichia coli to pulsed-field gel electrophoresis. The results suggest thatA. vinelandii chromosomes contain multiple identical chromosomes of about the same size as that ofE. coli.     

Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number

Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced. These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+). Similar induction was observed with the product of another plasmid gene, beta-lactamase. The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.     

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.     

Appearance of beta-lactamase activity in animal cells upon liposome-mediated gene transfer

A restriction fragment, 875 bp, which encodes for a beta-lactamase activity, was isolated from the Escherichia coli plasmid pBR322 DNA and entrapped in liposomes. The incubation of the DNA-liposomes with avian, murine, and human cultured cells results in the uptake of the DNA with the efficiency of around 2000 molecules per cell. Extracts of the recipient cells show a beta-lactamase activity as demonstrated by spectroscopic and microbiological methods. These results indicate the expression of a prokaryotic gene in eukaryotic cells.     

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

Nature of Col E1 Plasmid Replication in Escherichia coli in the Presence of Chloramphenicol

The colicinogenic factor E(1) (Col E(1)) in Escherichia coli continues to replicate by a semiconservative mechanism in the presence of chloramphenicol (CAP) for 10 to 15 hr, long after chromosomal deoxyribonucleic acid (DNA) synthesis has terminated. Following CAP addition, the rate of synthesis of plasmid DNA gradually increases to an extent dependent on the medium employed. Within 2 to 4 hr after the addition of CAP, replication in a glucose-Casamino Acids medium approaches a maximum rate representing approximately eight times an average rate which would be required for a net doubling of DNA per cell in one generation. The number of copies of Col E(1) DNA molecules that accumulate under these conditions approaches about 3,000 copies per cell, representing a 125-fold increase over the normal level of 24 copies per cell. The system is particularly convenient for studying the mechanism of DNA replication.     

Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell

A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene. DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region. No free copies of pOT have been detected. Both pOT and tk sequences may be amplified up to 20–200 copies of the SV40 early region. In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines. This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive. Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons. Transformation of E. coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E. coli. Excision is generally precise when pOT is introduced to the murine cells as a supercoiled molecule, and imprecise when pOT is introduced in linear form.     

Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans

The complexity of the genome of Micrococcus radiodurans was determined to be (2.0 +/- 0.3) X 10(9) daltons by DNA renaturation kinetics. The number of genome equivalents of DNA per cell was calculated from the complexity and the content of DNA. A lower limit of four genome equivalents per cell was approached with decreasing growth rate. Thus, no haploid stage appeared to be realized in this organism. The replication time was estimated from the kinetics and amount of residual DNA synthesis after inhibiting initiation of new rounds of replication. From this, the redundancy of terminal genetic markers was calculated to vary with growth rate from four to approximately eight copies per cell. All genetic material, including the least abundant, is thus multiply represented in each cell. The potential significance of the maintenance in each cell of multiple gene copies is discussed in relation to the extreme radiation resistance of M. radiodurans.     

Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization--RING-FISH

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.     

Amplification of bacterial plasmids without blocking protein biosynthesis

The effect of amino acids (presence or absence from the growth media) and metal ions on the replication of Escherichia coli plasmids in rel A+ strains was studied. It was found that: (i) The absence of one amino acid from the growth media had no effect on the plasmid copy number in prototrophic E. coli strains: (ii) The presence of only one amino acid in artificial media free of amino acids had a negligible effect on the plasmid copy number for the amino acids Ala, Arg, Glu, His, Leu, Phe, Thr, Trp, and Tyr: (iii) The combination of Met and Thr caused a rise in pBR322 plasmid copy number up to 90-100 plasmid copies per cell: (iv) The Fe3+ concentration had an amplification effect on E. coli plasmids. The pBR322 plasmid copy number for media free of amino acids and supplemented with 0.2-0.4 mM FeCl3 was 60-80 plasmid copies per cell: (v) The combination of Fe3+ with certain amino acids (Ala, Arg, Glu, Leu, Thr, and Trp) leads to a dramatic increase in the plasmid copy number reaching 180-270 plasmid copies per cell for the plasmid pBR322 and 20-24 for the plasmid pR100.     

Construction of a novel gene bank of Bacillus subtilis using a low copy number vector in Escherichia coli

Low copy number vector plasmid pCT571 was constructed to clone Bacillus subtilis genomic fragments in Escherichia coli. pCT571 confers KmR, TcR and CmR in E. coli and CmR in B. subtilis. It has unique restriction sites within the KmR and TcR markers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in E. coli. It also contains oriVRK2, which when supplied with the product of the trfA gene of RK2 in trans, allows pCT571 to replicate at 35-40 copies per chromosome equivalent. A B. subtilis gene bank was created by cloning partially Sau3A-digested and size-fractionated fragments of B. subtilis chromosomal DNA into the BamHI site of pCT571. DNA from 1097 KmR TcS transformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with EcoRI or EcoRI and SalI. Approximately 1000 hybrid plasmids were found with reasonably sized B. subtilis fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the B. subtilis chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in E. coli and B. subtilis. Hybrid plasmids which complement E. coli mutants for arg, his, lys, met, pdx, pyr and thr markers were identified from the gene bank. In B. subtilis the presence of argC, cysA, dal, hisA, ilvA, leuA, lys, metB, metC, phe, purA, purB, thr and trpC was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.     

Accumulation of human immunodeficiency virus type 1 DNA in T cells: results of multiple infection events.

Human immunodeficiency virus type 1 DNA synthesis was followed in a CD4+ line of T cells (C8166) grown in the presence or absence of a monoclonal antibody to CD4 that blocks infection By 48 h after infection, cultures grown in the presence of the antibody contained approximately 4 copies of human immunodeficiency virus type 1 DNA per cell, whereas those grown in the absence of the antibody contained approximately 80 copies of viral DNA per cell. Most of the viral DNA in cultures grown in the absence of the antibody was present in a broad smear of apparently incomplete viral sequences. In cultures grown in the presence or absence of the antibody, the 9.6-kilobase linear duplex of viral DNA appeared to undergo integration within 24 h of its appearance. These results demonstrate that T cells accumulate unintegrated human immunodeficiency virus type 1 DNA as a result of multiple virions entering cells.     

Unintegrated viral DNA sequences in a hamster tumor induced by bovine papilloma virus

A fibrosarcoma was induced in a hamster by bovine papilloma virus type 2 (BPV2). The content of BPV2 DNA sequences was measured by DNA-DNA and cRNA-DNA hybridizations. The tumor contained approximately 300 BPV2 genome equivalents per cell. Southern blot hybridization indicated that the viral DNA was in free form, the entire genome most likely being present. In situ hybridization with BPV2 cRNA showed that multiple genome copies were present in each cell. Neither virus particles nor virus coat antigens could be detected in the tumor. A cell line was established from the fibrosarcoma, and the cells contained multiple copies of the BPV2 genome. The latter was in free form, and all of the DNA sequences appeared to be present in multiple copies and in all cells. An extensive search failed to reveal the presence of virus or viral antigens.     

Transformation of Escherichia coli with DNA from Saccharomyces cerevisiae cell lysates

We developed a system to monitor the transfer of heterologous DNA from a genetically manipulated strain of Saccharomyces cerevisiae to Escherichia coli. This system is based on a yeast strain that carries multiple integrated copies of a pUC-derived plasmid. The bacterial sequences are maintained in the yeast genome by selectable markers for lactose utilization. Lysates of the yeast strain were used to transform E. coli. Transfer of DNA was measured by determining the number of ampicillin-resistant E. coli clones. Our results show that transmission of the Amp(r) gene to E. coli by genetic transformation, caused by DNA released from the yeast, occurs at a very low frequency (about 50 transformants per microg of DNA) under optimal conditions (a highly competent host strain and a highly efficient transformation procedure). These results suggest that under natural conditions, spontaneous transmission of chromosomal genes from genetically modified organisms is likely to be rare.     

Effect of the potential triplex DNA region on the in vitro expression of bacterial beta-lactamase gene in superhelical recombinant plasmids.

The effect of a pyrimidine/purine-biased stretch which has the potential to form an unusual triplex DNA structure on gene expression has been analyzed by measuring the activity of beta-lactamase as a reporter gene in recombinant plasmids. The Escherichia coli transformant carrying the plasmid p7ERS which has a potential triplex DNA region expressed about twofold more beta-lactamase activity than that carrying the plasmid pUC19. Since the expression of beta-lactamase has been shown to be affected by template topology in vitro, this in vivo observation suggests that the inserted pyrimidine/purine-biased stretch modulates the topology of flanking regions by forming unusual DNA structure to keep the template at the superhelicity favorable for the expression of beta-lactamase.