Donor Markers Research Articles

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.

On the question of the recombination frequencies of proximal markers in bacterial conjugation

A mathematical model of recombination is proposed, in which the probability of integration of leading donor markers into the recombinants P0 is different from zero (0 ≤ P0 ≤ 1). The experimental results are in good agreement with the model.

Effect of adenosine 5'-triphosphate-dependent deoxyribonuclease deficiency on properties and transformation of Haemophilus influenzae strains.

A transformation-deficient strain of Haemophilus influenzae, lacking adenosine 5'-triphosphate-dependent deoxyribonuclease activity, was isolated by selection for sensitivity to mitomycin. The mutant, designated JK57, possibily showed a moderate sensitivity to ultraviolet (UV) irradiation and treatment with methyl methane sulfonate. Contrary to the wild type, the mutant degraded chromosomal deoxyribonucleic acid (DNA) to some extent. However, after UV irradiation to the mutant degraded considerably less DNA than the wild type and the TD24 mutant of H. influenzae, the latter being equivalent to a recA mutant of Escherichia coli. A TD2457 double mutant, constructed by transferring the TD24 mutation into the JK57 strain, was as sensitive to deleterious agents and as deficient in transformation as the TD24 single mutant; in the double mutant, however, after UV irradiation chromosomal DNA was degraded to the same extent as in the JK57 mutant. The number of transformants per unit of radioactive donor DNA taken up by JK57 recipient cells was approximately 10-fold smaller than in the wild type. Presynaptically, the fate of donor DNA in the adenosine 5'-triphosphate-dependent deoxyribonuclease-deficient mutants was not different from that in the wild type. In contrast to TD24 and the TD2457 double mutant, in the JK57 mutant, recombinant-type activities (molecules carrying both the donor and recipient markers) were formed almost as well as in the wild type. After integration into the JK57 recipient genome, the rate of replication of the donor marker was equal to that of the recipient marker during a number of generations, which suggests that the donor DNA is normally integrated into the JK57 chromosome. It is suggested that transformed JK57 cells pass with a high frequency into a type of cells that can replicate their chromosomes many times but have lost the ability to form visible colonies after plating.

Mismatch correction in pneumococcal transformation: donor length and hex-dependent marker efficiency.

A hypothesis that preferential rejection of donor markers by the hex system of pneumococcus is due to lethal double-strand breaks has been examined in terms of its implications for the extent of the excision required. Experiments reported here were directed at asking whether hex-dependent marker efficiency depends on the length of the donor deoxyribonucleic acid (DNA). In the absence of intracellular competition for hex function, there was no detectable effect of DNA size on hex-dependent marker efficiency as donor DNA was sheared from greater than 1 x 107 daltons to 3.6 x 105 daltons. The latter DNA was purified by two successive velocity fractionations to ensure that the activity seen was representative of DNA of that size. Quantitative examination of the system shows that, for the lethal event hypothesis to be true, the excision step has to remove an average of 7,000 to 10,000 nucleotides. This figure is so much greater than that seen in other excision processes that alternate hypotheses should be considered. The presently known properties of the hex system can be accounted for by a model invoking the migratory features of type I restriction enzymes.

Chromosomal basis of the merozygosity in a partially deploid mutant of Pneumococcus.

A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

Recombinant clone heterogeneity in Escherichia coli conjunction: effect of pH and partially replicated recipient deoxyribonucleic acid.

At pH 6.8, a substantial fraction of recombinant colonies obtained from conjugation with an HfrH donor contained multiple recombinant classes in a single colony (polygenotype colony). In contrast, when the conjugation was performed at pH 7.6, the number of polygenotypic colonies was drastically reduced, and the recombinant colonies were predominantly monogenotypic or digenotypic. Genetic analysis revealed that the digenotypic recombinants differ in those donor markers near the origin of DNA replication but share those donor markers near the terminus. This integration pattern suggests that the formation of digenotypic recombinants involves recombination of a single copy of the exogenome with a partially replicated recipient DNA molecule. This suggestion was supported by examination of the genotype of recombinant colonies recovered from crosses with an HfrKL96 donor which was derived from HfrH but transfers its chromosome in the reverse direction.

Chromosome transfer in Proteus mirabilis mediated by hybrid plasmid.

A previously-described fused plasmid, P-lacRIdrd19, was found to mediate chromosomal transfer between cells of Proteus mirabilis strain PM5006; PM5006-(P-lacRIdrd19) was usually the donor and various auxotrophs of PM5006 resistant to nalidixic acid and/or streptomycin were recipients. The donor was usually counterselected with nalidixic acid and/or high concentrations of streptomycin. Recombination experiments with single markers indicated a 40-fold variation in recombination frequencies for different markers. Mapping double-auxotrophic markers by their gradient of transmission confirmed this variation and placed each of two independent isolates of eight markers in a linkage group his-ser-ura-pyrB-trp-cys-ade-ilv. Some donor markers did not register. Despite low recombination frequencies, interrupted mating experiments showed a polarity of early marker transfer. The segregation of unselected markers confirmed the order of some markers and showed that genetic material passed from the presumptive donor to the recipient. Recipients with two auxotrophic markers which could not be cotransduced by phage 5006M were converted to prototrophy by conjugation. The plasmid transferred to recipients at high frequency and all recombinants carried it. Recombinants could act as donors in further matings. Recombinants were fully susceptible to phage 5006M, unlike transductants of PM5006 by this phage. Direct involvement of the plasmid was indicated by drastically diminished recombination frequencies in crosses with recipients carrying P-lac as resident. P-lac had previously been shown to reduce the frequency of transfer of the hybrid plasmid to cells harbouring it. The histidine region was the first to register in recipients and recombined at the highest frequency of 5 times 10-6/donor cell. Some temporary association of plasmid and perhaps only the histidine region of the chromosome is favoured as the mechanism of chromosomal transfer. This could explain why not all donor markers could be mapped. Transduction and transformation were excluded as the cause of results.

Marker rescue from UV-irradiated bacteriophages: A UV-sensitive region in bacteriophage T2

Marker rescue was studied with T2 and T4 strains as irradiated donor strains and T2 am 39 rIIA or T2 am 47 gt 29 am 55 as helper strains. After selection of one marker from the donor strain, the frequency of the second donor marker was determined (corescue frequency). Corescue frequencies decreased with increasing doses of UV irradiation. In the region am 47- am 55 there was no difference between frequencies of corescue from T2 and T4 as donor strains. In the region am 39- rIIA , frequencies of corescue from T2 were much lower than from T4 and could be ascribed to rescue of the two markers by independent rescue events. To explain the difference between T2 and T4, the following possibilities were investigated: (1) protection of a UV-sensitive site in T4 by a T4 gene product; (2) influence of partial exclusion of helper strain T2 by T4, and (3) a difference between T2 and T4 with respect to UV sensitivity in the region am 39- rIIA . The first possibility could be rejected. Exclusion might play a minor role in corescue from T4. Probably, the region am 39- rIIA in T2 is more susceptible to UV damage than in T4, presumably owing to a higher content of potential thymine dimers. The mean lengths of rescued DNA fragments at various doses of UV irradiation were estimated by using the observed corescue frequencies. Extrapolation of the relation between UV dose and length of rescued DNA fragments predicts a UV dose of 360–530 PLH required for rescue of DNA fragments of about two map units in the region of T4 gene α gt .

Marker rescue from UV-irradiated bacteriophages: A UV-sensitive region in bacteriophage T2

Marker rescue was studied with T2 and T4 strains as irradiated donor strains and T2 am39 rIIA or T2 am47 gt29 am55 as helper strains. After selection of one marker from the donor strain, the frequency of the second donor marker was determined (corescue frequency). Corescue frequencies decreased with increasing doses of UV irradiation. In the region am47- am55 there was no difference between frequencies of corescue from T2 and T4 as donor strains. In the region am39- rIIA, frequencies of corescue from T2 were much lower than from T4 and could be ascribed to rescue of the two markers by independent rescue events. To explain the difference between T2 and T4, the following possibilities were investigated: (1) protection of a UV-sensitive site in T4 by a T4 gene product; (2) influence of partial exclusion of helper strain T2 by T4, and (3) a difference between T2 and T4 with respect to UV sensitivity in the region am39- rIIA. The first possibility could be rejected. Exclusion might play a minor role in corescue from T4. Probably, the region am39- rIIA in T2 is more susceptible to UV damage than in T4, presumably owing to a higher content of potential thymine dimers. The mean lengths of rescued DNA fragments at various doses of UV irradiation were estimated by using the observed corescue frequencies. Extrapolation of the relation between UV dose and length of rescued DNA fragments predicts a UV dose of 360–530 PLH required for rescue of DNA fragments of about two map units in the region of T4 gene α gt.

Destruction of low efficiency markers is a slow process occurring at a heteroduplex stage of transformation.

Direct evidence is presented that the mechanism which discriminates against low efficiency markers in transformation of Diplococcus pneumoniae of genotype hex+ acts on them after the formation of donor-recipient heteroduplexes. This conclusion is based on assays of the transforming activity of donor markers in lysates made after various times of incubation of recipient cells following exposure to DNA. The activity of a low efficiency marker rises substantially, indicating formation of native-like heteroduplex structures, and then falls. At 37° C the process is essentially completed 10 minutes after entry, and the apparent half life of a susceptible heteroduplex is 1.5 to 2 minutes. Data from these and other experiments imply that about as many of the surviving low efficiency markers have simply escaped attack as have been inserted into both strands by the excision-repair process suggested by Ephrussi-Taylor.

Fate of donor deoxyribonucleic acid in a highly transformation-deficient strain of Haemophilus influenzae.

A transformation-deficient strain of Haemophilus influenzae (efficiency of transformation 10(4)-fold less than that of the wild type), designated TD24, was isolated by selection for sensitivity to mitomycin C. In its properties the mutant was equivalent to recA type mutants of Escherichia coli. The TD24 mutation was linked with the str-r marker (about 30%) and only weakly linked with the nov-r2.5 marker. The uptake of donor deoxyribonucleic acid (DNA) was normal in the TD24 strain, but no molecules with recombinant-type activity (molecules carrying both the donor and the resident marker) were formed. In the mutant the intracellular presynaptic fate of the donor DNA was the same as that in the transformation-proficient (wild-type) strain, and the radioactive label of the donor DNA associated covalently with the recipient chromosome in about the same quantity as in the wild type. However, many fewer donor atoms were associated with segments of the mutant's recipient chromosome as compared with segments of the wild-type chromosome. In the mutant the association was accompanied by complete loss of donor marker activity. The lack of donor marker activity of the donor-recipient complex of DNA isolated from the mutant was not due to lack of uptake of the complex by the second recipient and its inability to associate with the second recipient's chromosome. Because the number of donor-atom-carrying resident molecules was higher than could be accounted for by the lengths of presynaptic donor molecules, we favor the idea that the association of donor DNA atoms with the mutant chromosome results from local DNA synthesis rather than from dispersive integration of donor DNA by recombination.

Chromosomal integration of Klebsiella nitrogen fixation genes in Escherichia coli.

SUMMARY Escherichia coli, c-m7, a His+Nif+ hybrid obtained by intergeneric mating with a Klebsiella pneumoniae donor strain, also inherited the unselected markers gnd and rfb. The R factor, R144drd3, which had been used to confer fertility on the donor, was present, detectable as covalently closed circular DNA of molecular weight 69 × 106 daltons. No other species of supercoiled DNA were isolated and the elimination of R144drd3 did not result in the loss of Klebsiella genes. Segregation analysis of donor markers indicated that the Klebsiella DNA was integrated at the his region of the E. coli chromosome in the probable order his-gnd-nif-rfb. Strain c-m7 produced a nitrogenase physiologically identical to that of K. pneumoniae, but synthesized a heteromeric species of gluconate-6-phosphate dehydrogenase.

A plasmid of Streptomyces coelicolor carrying a chromosomal locus and its inter-specific transfer.

SUMMARY: A strain of Streptomyces coelicolor a3(2) was selected in which the wild-type autonomous SCPi plasmid had acquired an insertion of a region of the host chromosome bearing the cysB locus. This strain, called an SCPi' strain by analogy with F' strains of Escherichia coli, donated the cysB + allele very efficiently to a cysB recipient. The Cys+ progeny of such crosses were heterozygotes that gave rise to Cys- segregants by plasmid loss. Other donor markers, not carried on the plasmid, were donated with much lower frequencies. The SCPi-cysB plasmid was also donated rather efficiently to a strain of Streptomyces lividans, previously shown to receive the wild-type SCPi plasmid. Transfer back from S. lividans to S. coelicolor a3(2) occurred at a very much lower frequency.

Poor transformability with nov r and ery r donor DNA of some mitomycin-C-sensitive strains of Haemophilus influenzae

Three mitomycin-C-sensitive (MC s) strains of Haemophilus influenzae, being poorly transformable with DNA carrying the antibiotic resistence markers nov r and ery r, were further investigated to determine the cause of their poor transformability. After being genetically integrated into the mutant-recipient genome the donor marker is replicated at the same rate as in the wild type, indicating that recombination in the mutant strains is normal. In the mutants, designated T d (transformation-deficient), the poor transformability for the nov r and ery r markers is due to the lack of phenotypic expression of the markers, because the strains are killed by concentrations of antibiotics normally used to select for nov r and ery r transformants. Since the strains exhibit extreme sensitivity both to deoxycholate and osmotic shock in the presence of EDTA, the increased sensitivity to antibiotics (including mitomycin-C) is probably caused by a change in the cell envelope. Although recombination in the mutant strains proceeds normally, the T d mutation nevertheless decreases both the rate of inactivation and of integration of donor DNA.

Mapping the chromosome of Yersinia pseudotuberculosis by interrupted mating.

SUMMARY: By interrupting matings at different times, the sequential transfer of selected and unselected markers to recipient bacteria was followed in Yersinia pseudotuberculosis. Under the best mating conditions employed, the earliest marker was transferred within 13 min and the latest within 98 min of impinging mating mixtures on membranes. All tested donor markers appeared in recombinants. A genetic map based on the entry times of markers indicated a single linkage group showing much similarity to that of Escherichia coli. The high frequency donor transferred markers in a clockwise direction, its origin corresponding to a point near 60 min on the E. coli map.

Transformation in Bacillus subtilis. Fate of newly introduced transforming DNA.

Donor deoxyribonucleic acid (molecular weight 5-8×107) introduced into competent cells of Bacillus subtilis is converted to molecules with a weight average molecular weight of ≅9×106. These molecules, having little transforming activity, constitute in all probability eclipse phase DNA. At least part of the DNA is transiently complexed with a cellular component, changing its buoyant behaviour in CsCl gradients. When shortly after uptake of donor DNA the total DNA extracted from recipient cells is sheared to a molecular weight of ≅8×106 or less, no eclipse phase is discernable. Donor marker frequencies in sheared, reisolated DNA mixtures decrease by a factor of 4 as a function of time of incubation of the transforming cells. This indicates that only 25% of the irreversibly absorbed DNA is finally integrated into the recipient genome.

Molecular events accompanying the fixation of genetic information in Haemophilus heterospecific transformation.

Heterospecific transformation between Haemophilus influenzae and H. parainfluenzae was investigated by isopycnic analysis of deoxyribonucleic acid (DNA) extracts of (3)H-labeled transforming cells that had been exposed to (32)P-labeled, heavy transforming DNA. The density distribution of genetic markers from the resident DNA and from the donor DNA was determined by transformation assay of fractions from CsCl gradients, both species being used as recipients. About 50% of the (32)P atoms in H. parainfluenzae donor DNA taken up by H. influenzae cells were transferred to resident DNA, and only a small amount of the label was lost under conditions of little cell growth. There was less transfer in the reciprocal cross, and almost half of the donor label was lost. In both crosses, the transferred donor material transformed for the donor marker considerably more efficiently when assayed on the donor species than on the recipient species, indicating that at least some of the associated (32)P atoms are contained in relatively long stretches of donor DNA. When the transformed cultures were incubated under growth conditions, the donor marker associated with recipient DNA transformed the donor species with progressively decreasing efficiency. The data indicate that the low heterospecific transformation between H. influenzae and H. parainfluenzae may be due partly to events occurring before association of donor and resident DNA but results mostly from events that occur after the association of the two DNA preparations.

Kinetics of Integration of Transforming DNA in Pneumococcus

Integration of donor genes, as measured by recovery of their transforming activity from eclipse in lysates of newly transformed cells of pneumococcus, has been followed at temperatures from 0 to 40 degrees . There is a lag in the recovery curve that is marker-dependent and increases as temperature falls. An Arrhenius plot of the rates shows a sharp break between 15 and 20 degrees . Brief exposure of the system to 37 degrees before incubation at 10 or 15 degrees removes the lag and raises the subsequent rate of recovery. This activation is unstable, however, and disappears when the cells are held at 0 degrees after the exposure at 37 degrees and before incubation at 15 degrees . The results are interpreted in terms of a reaction sequence A right harpoon over left harpoon B --> C, with activation energies for the first forward rate-constant of the order of 50 Cal/mol, for the second, 20-21 Cal/mol, and for the reverse reaction, less than 20 Cal/mol. The properties of the first step, including its marker dependence, are the same as those observed earlier for stabilization of donor markers against intracellular inactivation, and it is suggested they may reflect an activation of the recipient chromosome prerequisite to synapsis.

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com(-56), was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process.

Ultraviolet inactivation and excision-repair in Bacillus subtilis IV. Integration and repair of ultraviolet-inactivated transforming DNA

The fate of UV-irradiated transforming Bacillus subtilis DNA after uptake by competent cells was investigated by assaying the reextracted donor DNA on uvr + and uvr − recipients. This procedure enables the detection of recombinant type molecules containing dark-repairable damage. The results allow the following conclusions: 1. ( i) transforming DNA irradiated with 2000 erg/mm 2 of UV is integrated as efficiently, or nearly as efficiently, as non-irradiated DNA. Under these conditions damage in the integrated molecules accounts almost completely for the UV inactivation of transforming DNA; 2. ( ii) after irradiation with 20 000 erg/mm 2 of UV a certain degree of selective exclusion of damaged DNA molecules from the integration process occurs. The exclusion accounts for approximately 10% of total inactivation of the donor marker activity in uvr + and for less than 1% in uvr − recipients; 3. ( iii) in uvr + recipeints repair of UV lesions in transforming DNA starts after integration and is completed within approximately 45 min with DNA irradiated with 2000 erg/mm 2 of UV, and within approximately 2 h after a UV dose of 15 000 erg/mm 2; 4. ( iv) eclipse phase DNA extracted from uvr + or uvr − first recipients is not dark-repaired by uvr + second recipients.