Fate of Transforming Deoxyribonucleate in Bacillus subtilis

The majority of donor deoxyribonucleate (DNA) at early stages after uptake was found in a complex with a cell component which changes its buoyant behavior on equilibrium density gradients. Analysis of the recipient cell lysates, after treatment to dissociate the complex, showed about two-thirds of the donor molecules in denatured form and the rest associated with recipient DNA. Incubation of cells after DNA uptake leads to the disappearance of denatured donor DNA and to the increase of donor label associated with recipient DNA. Some characteristics of a component from intact cells or spheroplasts with affinity for denatured Bacillus subtilis DNA are described.     

The segregation of DNA in epithelial stem cells

It has recently been suggested that stem cells may invariably keep, from one division to the next, the daughter DNA molecules that contain the older of the two parental strands—that is, they may retain a complete set of “immortal strands,” through successive cell divisions (Cairns, 1975). We can test this hypothesis by labeling either the old immortal strands at the time the stem cells are created or the newly synthesized strands during subsequent divisions of the stem cells. In the former case, the stem cells should become permanently labeled; in the latter case, they should eliminate their label on their second division.Experiments of this sort have been conducted with the tongue papilla under steady state conditions and with the regenerating small intestinal crypts. The results clearly show that by far most of the multiplying cells in tongue and intestinal epithelium segregate their DNA “randomly” at mitosis. Nevertheless, the results, though far from conclusive, suggest that there are a small number of cells (1–5 in the stem cell region of each crypt and one at the base of each column of cells in the tongue) that selectively segregate their old and new DNA strands in the expected way. Thus in the immortal strand labeling experiments, there are a few labeled cells that retain their label for up to 4 weeks; conversely, in the new strand labeling experiments, a few cells appear to rid themselves of label after intervals equivalent to approximately two cell cycles.     

Number of Deoxyribonucleic Acid Uptake Sites in Competent Cells of Bacillus subtilis

Two direct methods are presented for estimating the average number of deoxyribonucleic acid (DNA) uptake sites in competent cells of Bacillus subtilis from measurement of (14)C- or (3)H-thymine-labeled DNA uptake by competent culture. Advantage is taken of two facts: (i) effective contact between competent cells and transforming DNA molecules is established within a short time after mixing them together, and (ii) DNA molecules enter the competent B. subtilis cells in a linear fashion at a finite speed. From the number of DNA molecules initially attached to competent cells by brief exposure to transforming DNA in the first method or from the rate of DNA uptake by competent culture in the second method, the average number of DNA uptake sites is calculated to be 20 to 53 per competent cell.     

The synthesis and distribution of DNA in the macronucleus of Euplotes eurystomus

In an attempt to recognize any ordering of DNA synthesis which might occur in ciliates, the distribution of labelled DNA in the G₁ and S periods of the first, second, and third generations of a synchronized population of Euplotes eurystomus was studied by means of autoradiography. The results presented here show that the replicating DNA which at the time of label incorporation is restricted to a morphologically identifiable narrow region of the nucleus (the replication band), becomes dispersed and is evenly distributed throughout the nucleus. This dispersal of labelled DNA occurs previous to division and is observable throughout the G₁ period of the following generation. During the S phase of this second generation, this previously labelled DNA once again becomes restricted to a small portion of the nucleus. Now, however, it is present at the tips of the macronucleus independently of the position of the replication band. Again the labelled DNA is found to be dispersed during G₁ of the third generation. In the S period of this third generation however the radioactive DNA again appears localized in the replication bands which are found at the same position in the nucleus where they were when the pulse was given two generations earlier. The observations derived from the autoradiographic analysis suggest a non-permanent organization for at least those DNA molecules which are replicated during the first third of the S period. This DNA can be associated in either of two specific locations, replication band or tip, and these two patterns of organization alternate from generation to generation.     

Do trophectoderm and inner cell mass cells in the mouse blastocyst maintain discrete lineages?

The extent to which trophectoderm (TE) and inner cell mass (ICM) lineages in the mouse blastocyst remain distinct during the period from the commencement of cavitation up until 48 h later in culture was investigated. Fluorescent latex microparticles were used to label exclusively all TE cells in nascent blastocysts and the position of labelled progeny in cultured blastocysts was examined by disaggregation, by serial sectioning and by whole-mount analyses. The results indicate that, in most blastocysts (80-90%), TE and ICM lineages are entirely separate during this period while in the remainder lineage crossing is limited usually to only one or two cells of either tissue.     

Cell surface glycoproteins of CHO cells. II. Surface distribution and pathway of internalization

The surface distribution and pathway for internalization of the major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent trinitrobenzenesulfonate. Molecules, haptenized with trinitrophenol groups, the majority of which are in a group of high molecular weight acidic glycoproteins (HMWAG), were labelled at 4 degrees C with anti-dinitrophenol immunoglobulins coupled to fluorescein isothiocyanate (FITC), horseradish peroxidase, or colloidal gold and either immediately fixed for mapping their distribution or followed intracellularly after warming to allow endocytosis to proceed. The distribution of label on the CHO cell surface was non-random with a large proportion arranged in clusters from 100 to 300 nm in diameter. Antibody label was concentrated heavily on microvilli, and about 10% of the molecules were always associated with clathrin-coated pits. Upon warming the cells to 37 degrees C, HMWAG were internalized immediately into smooth-membraned tubules (less than 80 nm luminal diameter) that appeared to connect with vesicles (less than 300 nm luminal diameter) located in the cortical cytoplasm. By 60 min, labelled antibody was located within larger vesicles (greater than 300 nm luminal diameter) that had a morphology characteristic of multivesicular bodies and not lysosomes. There was no evidence for entry of labelled molecules into either electron-dense, secondary lysosomes or into the Golgi cisternae, suggesting that neither compartment is involved in the major pathway of cell surface endocytosis. Our results are consistent with the view that the majority of plasma membrane protein are internalized as small discrete domains by a pathway very similar to that described by others for adsorptive endocytosis.     

The mode of action of cytotoxic and antitumor 1-nitroacridines. III. In vivo interstrand cross-linking of DNA of mammalian or bacterial cells by 1-nitroacridines

To define a critical lesion in presumable target DNA cause in vivo by the antitumor and cytotoxic 1-nitroacridines, Ehrlich ascites tumor (Eat) cells implanted into mice, HeLa cells grown in monolayer culture or Bacillus subtilis SB 1058 strain cells were exposed to Ledakrin [Nitracrine; 1-nitro-9-(3'-dimethylamino-n-propylamino)acridine], its non-antitumor congeners, or mitomycin C tested for comparison; total intracellular DNA was isolated from control or treated cells and denatured by heat, alkali or formamide, after which the chemically-induced fraction of interstrand cross-linked DNA molecules was assessed by thermal denaturation-renaturation curve analysis, hydroxylapatite column chromatography, or partitioning in a Dextran T500-polyethylene glycol 6000 biphasic system. Ledakrin, as compared to mitomycin C, was a very effective cross-linking agent, inducing one covalent cross-link per approx. 20 X 10(3) (B. subtilis), 56 X 10(3) (HeLa) or 80 X 10(3) (Eat) DNA base pairs. The first cross-links were introduced in B. subtilis cell genomes at minimal bactericidal concentrations of Ledakrin of mitomycin C. Ledakrin failed to induce discernible cross-linking of bihelical DNA when complexed with in cell-free system. Unlike the other two 1-nitroacridines which cross-linked DNA of cultured HeLa or B. subtilis cells, the non-antitumor 2-, 3- or 4-nitroacridines did not cause such effect and diminished cross-linking by Ledakrin or mitomycin C. Hence, upon metabolic activation in mammalian or bacterial cell Ledakrin and, most probably other 1-nitroacridines, become very effective DNA cross-linking agents and such effects appear to be responsible for the antitumor and potent cytotoxic activities of 1-nitroacridines.     

Time-resolved fluorometry: a sensitive method to quantify DNA-hybrids.

Europium and other lanthanides can be excitated with UV-radiation, whereafter the energy is released as fluorescence, delayed in time up to 1 ms after the excitation. Eu can be used as a sensitive label in biological assays. Here we report on the application of time-resolved fluorometry to detect nucleic acid hybrids. The probe DNA was tagged with a hapten, either a fluorene or a sulfone group. After hybridization the probe DNA was detected by a two-step immunological assay with the second antibody labelled with Eu. The method is quantitative with a detection limit of 0.3 pg of actual target regions of immobilized adenovirus genomic DNA. The label was also used in sandwich hybridization, which allowed analyzing nasopharyngeal mucus for the presence of adenovirus.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Fate of heterologous deoxyribonucleic acid in Bacillus subtilis.

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thy minus trp minus. Shortly after uptake, most of the radioactive Escherichia coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with Escherichia coli 2-H and 3-H-labeled DNA. Association of radioactivity with recipient DNA decreased to one-fourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the Escherichia coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

Recombinational-type transfer of viral DNA during bacteriophage 2C replication in Bacillus subtilis.

The Bacillus subtilis phage 2C contains one molecule of double-stranded DNA of about 100 x 10(6) daltons in which thymine is replaced by hydroxymethyluracil; the two strands have different buoyant densities. Parental DNA, labeled with either [3H]uracil of [32P]phosphate, was quite effectively transferred to offspring phage, and the efficiency of transfer was the same for the two strands. Labeled nucleotide compositions of the H and L strands from parental and progeny virions were very close. These data exclude a degradation of the infecting DNA and reutilization of nucleotides. Upon infection of light unlabeled cells with heavy radioactive viruses, no DNA with either heavy or hybrid density was extracted from offspring phage. Instead, an heterogeneous population of DNA molecules of densities ranging from that of almost hybrid to that of fully light species was obtained. Shear degradation of such progeny DNA to fragments of decreasing molecular weight produced a progressive shift to the density of hybrid molecules. Denaturation of sheared DNA segments caused the appearance of labeled and heavy single-stranded segments. These findings indicate that 2C DNA replicates semiconservatively and then undergoes extensive genetic recombination with newly formed viral DNA molecules within the vegatative pool, thus mimicking a dispersive transfer of the infecting viral genome. The pieces of transferred parental DNA have an average size of 10 x 10(6) daltons.     

A universally applicable process for preparing stoichiometrically 1:1 labelled functional proteins

A universally applicable labelling and purification process was established to prepare biologically active proteins with a stoichiometric 1:1 ratio of attached dye-label. The dye-label is linked to a specific DNA sequence, which acts as a barcode-like tag for affinity purification. The DNA-dye tag is covalently bound to the target protein, which is present in excess to assure the binding of not more than one dye per molecule. Affinity purification occurs at magnetic beads that are functionalized with oligonucleotides that are complementary to the DNA-tag of the labelled proteins but for one or two mismatches. Washing removes all unbound, unlabelled molecules. The labelled protein is subsequently released by the addition of a fully complementary oligonucleotide. This process allows a gentle purification of a protein fraction that has exactly one label attached to each molecule under conditions that preserve protein structure.     

Polarity of DNA entry in transformation of Streptococcus pneumoniae

Summary DNA transport in Streptococcus pneumoniae was studied using donor molecules labelled either at the 3 or at the 5 end, on one strand only. In contrast to 5 end label, 3 end label was not taken up by the cells indicating that entry is a polarized process. Our results together with those of previous studies are consistent with a model for entry in which double-stranded donor DNA is nicked on binding at the cell surface. Entry of a single strand then proceeds linearly from a newly formed 3 end to the extremity of the donor fragment.     

Programmierter Zelltod bei Bakterien

Programmed cell death (PCD) in bacteria In bacteria, cell death occurs under certain stressful conditions, and this process has been designated as programmed cell death (PCD). The biological basis of the PCD are two molecules, a stable toxin protein and an unstable antitoxin being either a short RNA molecule or a protein. The antitoxin has to be synthesized permanently to neutralize the toxin. Both components form a TA module. When the synthesis of the antitoxin is blocked or when it is degraded completely, the free toxin acts either bacteriostatic or bactericide. So far, five different mechanisms have been described of how the antitoxin neutralizes the toxin in the absence of stress. Sporulating Bacillus subtilis cells exert cannibalism that means they kill and lyse non‐sporulating cells to take up their nutrients. Streptococcus pneumoniae cells can carry out fratricide. They kill and lyse neighbouring cells, take up fragments of their chromosomal DNA and recombine them with their own DNA. This can result in the uptake of new genes. At the end, two examples of application of TA modules in biotechnology are described.     

A cell-free system for the replication fo bacteriophage M-13 duplex DNA.

Cell-free extracts from M-13 am5 infected Escherichia coli cells which are highly concentrated on cellophane membrane disks replicate efficiently endogenous M-13 duplex DNA. If the reaction is carried out in the presence of bromodeoxyuridine triphosphate, the majority of the label is found in two classes of hybrid DNA molecules in which either the viral or the complementary strand is newly synthesized. A minor portion of the label is incorporated into fully synthetic duplex DNA. DNA synthesis requires ATP and is inhibited by nalidixic acid, novobiocin, and arabinosylnucleoside triphosphates. Rifampicin blocks preferentially the synthesis of molecules with labeled complementary strands. A similar effect is observed upon addition of the helix-destabilising M-13 gene V protein. In contrast, addition of E. coli helix-destabilising protein (Eco HD-protein) stimulates the synthesis of both types of hybrid DNA molecules as well as the formation of fully synthetic duplex DNA.     

Epidermal cell proliferation. I. Changes with time in the proportion of isolated, paired and clustered labelled cells in sheets of murine epidermis

A new technical approach to analysing labelled cells in sheets of epidermis is presented. The changes in the proportion of isolated single labelled cells, paired or clusters of 3, 4, or more than 4, labelled cells in sheets of epidermis from the back of the mouse have been analysed at various times up to 500 h after 3HTdR administration at either 03.00 h or 15.00 h. The technique is not dependent on the relative number of labelled cells (i.e. the labelling index) but on the spatial distribution of labelled cells. The data cannot be adequately explained on the basis of a simple homogeneous stem cell population in the basal layer but can be better understood on the basis of an hierarchical stem cell-dividing transit proliferative model. The data are consistent with an average cell cycle time of about 100 h but there are suggestions of considerable cell kinetic heterogeneity. The data also suggest that the amount of lateral cell movement within the basal layer is small. The results may suggest that some stem cells either loose label in a manner similar to that suggested by Cairns (1975) i.e. through a process of selective segregation of their DNA strands, or that they have an extremely short S phase duration as postulated earlier (Potten et al. 1982). The present data have been extensively mathematically modelled in an accompanying paper. The model which best fits all the data is an hierarchical scheme with three cell divisions in the transit population but some branches of the lineage may be prematurely terminated by the early production of post-mitotic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

Observations regarding DNA replication sites in human cells in vivo following infusions of iododeoxyuridine and bromodeoxyuridine

In studies using bromodeoxyuridine (BrdUrd) and/or iododeoxyuridine (IdUrd) to label S phase cells in cancer patients, several unique observations were made regarding DNA replication sites and the organization of newly synthesized DNA in post-mitotic cells. While the majority of tumour specimens removed at the end of infusions demonstrated concentration of replication sites around the nuclear membrane, biopsies obtained in leukaemic patients 1 week later demonstrated several distinct patterns of labelling. For example, one, two or all lobes of granulocytes were labelled. Scavenger macrophages bearing labelled leukaemic cells in their cytoplasm were also seen. Sequential IdUrd/BrdUrd labelling of solid tumours showed various patterns of nuclear/nucleolar/membrane labelling, allowing more precise localization of early versus late replication sites.     

Uptake of CdSe and CdSe/ZnS quantum dots into bacteria via purine-dependent mechanisms

Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type Bacillus subtilis incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In Escherichia coli, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light.