Low Efficiency Markers Research Articles

Mismatch repair in Streptococcus pneumoniae: relationship between base mismatches and transformation efficiencies.

Genetic transformation in Streptococcus pneumoniae involves the insertion of single-stranded pieces of donor DNA into a recipient genome. Efficiencies of transformation strongly depend on the mutations (markers) carried by donor DNA. Markers are classified according to their transforming efficiencies into very high, high, intermediate, and low efficiency. The last is approximately 1/20th as efficient as the first. This marker effect is under the control of the Hex system, which is thought to correct mismatches at the donor-recipient heteroduplex stage in transformation. To investigate this effect, wild type, mutant, and revertant DNA sequences at five genetic sites within the amiA locus were determined. The results show that low-efficiency markers arise from transitional changes A . T to G . C. The transversion A . T to T . A corresponds to an intermediate-efficiency marker. Transversions G . C to T . A and G . C to C . G lead to high-efficiency markers. Among the eight possible mismatches that could exist transiently at the heteroduplex stage in transformation, only two--namely, A/G and C/C--are not corrected by the Hex system. It is noteworthy that the four possible base pairs (A . T, T . A, G . C, and C . G) have been encountered at the very same site (amiA6 site), which constitutes a good illustration of the marker effect. DNA sequence analysis also reveals that short deletions (33 or 34 bases long) are integrated with very high efficiencies. These results confirm that the Hex system corrects point mismatches harbored in donor-recipient heteroduplexes thousands of bases long. The correction pattern of the Hex system toward multiple-base mismatches has also been investigated. Its behavior toward double-base mismatches is complex, suggesting that neighboring sequences may affect the detection of mispaired bases.

Excision and repair of mismatched base pairs in transformation of Streptococcus pneumoniae.

The use of heteroduplex DNA molecules as donors in pneumococcal transformation makes it possible to follow the fate of each DNA strand. The integration efficiency of each strand depends strongly upon the single base changes it carries. The function (hex) which reduces drastically the transformation yield of markers referred to as low efficiency (LE) tends to remove either donor strand without respect ot which one is introduced. In the case of high efficiency (HE) markers the reduction in the transformation yield involves the elimination of only one donor strand. For a given locus it can be either one depending upon the mutation. The reduction in transformation yield can be less drastic for HE markers than for both strands of the LE markers. These data are discussed in terms of differences in the affinity for mismatched base pairs. We have studied the transfer of information from each donor DNA strand to the recipient genome, on the basis of differences in the rates of phenotypic expression of a given marker introduced on opposite strands. Results show that, as in the case of LE markers, the information from HE markers, when introduced on the strand recognized by the hex function, is transmitted to both strands of the recipient molecule. Correction of the recipient strand to homozygosis probably accounts for this information transfer. These results, together with earlier investigations, strongly suggest that the hex function is an excision-repair system acting on donor-recipient base pair mismatches.

A hex mutant of Haemophilus influenzae

A mutant of Haemophilus influenzae which does not discriminate between low efficiency (LE) and high efficiency (HE) markers has been isolated. The mutant does not differ wild type in its sensitivity to ultraviolet radiation, methyl methanesulfonate (MMS) mitomycin C, and nitrous acid. Spontaneous mutation frequencies for three loci studied are 10- to 30-fold higher in the mutant than in the wild type strain. Low- and high-efficiency transforming markers are equally UV-resistant when assayed on this mutant. This mutant is thus similar to the hex mutant of Streptococcus pneumoniae.

Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation.

The efficiency of transformation of point mutations depends upon base pair mismatches during the recombination process. For low-efficiency markers, the genetic information carried on the donor deoxyribonucleic acid is preferentially lost. To understand this elimination process, we investigated the effect of the size of donor deoxyribonucleic acid on the relative efficiency of low-efficiency point mutations. The deoxyribonucleic acid was shortened either by mechanical shearing or by restriction enzyme treatments. The results indicate that transformation by low-efficiency markers was not affected by shortening the distance between them and the end of the molecule any more than was transformation by the other markers. Moreover, no lethal event could be detected for either cell or chromosomal marker survival. These data do not exclude the double-strand-break hypothesis that was proposed to explain the loss of genetic information for low-efficiency markers, but they offer no support for it.

A transforming marker that produces merodiploids with high efficiency and stable transformants with low efficiency in Streptococcus.

A mutation (ery-r8) conferring a high level of resistance to erythromycin in the Challis strain of Streptoccus sanguis can be transferred to wild-type erythromycin-sensitive recipients via single molecules of donor DNA. The transformants thus produced are of two types: (1) cells slightly more resistant to erythromycin than wild-type and capable of segregating (at a frequency of 2 X 10(-4)/bacterium/generation) either wild-type or highly-resistant cells like the original donor type; (2) cells phenotypically and genotypically identical to the original donor type. The unstable diploids (ery-r8/+) occur with a frequency equivalent to that obtained with high-efficiency (HE) markers, whereas the stable donor-type (ery-r8) transformants occur with about five hundred times lower frequency. Penetration of the wild-type recipient by more than one molecule of DNA bearing the ery-r8 marker increases by as much as seven times the incidence of stable transformants. UV-irradiation of molecules bearing the ery-r8 marker diminishes their ability to cooperate in producing a stable transformant, although the UV sensitivity of stable transformant production by a single DNA molecule is not different from that of diploid production. Hence, stable transformants do not appear to be produced by a process typical of low efficiency (LE) markers, which are generally highly sensitive to ultraviolet irradiation. Moreover, stable ery-r8 transformants are produced with equally low frequencies in strains of S. pneumoniae that discriminate (hex+) and fail to discriminate (hex--) between HE and LE markers. We postulate that all transformations by the ery-r8 marker result in ery-r8/+ diploids, and that segregation results in the infrequent stable transformants of the original donor type. This hypothesis is supported by the observations that rifampin treatment of ery-r8/+ populations increases the frequency of segregation and similar treatment of wild-type recipients under-going transformation by the ery-r8 marker increases the frequency of stable transformants.--In producing the ery-r8/+ transformant the r8 allele is integrated close to the site of its wild-type homolog, since single molecules of DNA from this transformant can be shown to carry both alleles. Segregation of either the ery-r8 or + allele is not detectably enhanced by acridine orange or thymidine deprivation.--The ery-r8 marker occurs close to a site of mutation (ery-r2) which confers erythromycin resistance upon ribosomes. When the r2 and r8 markers are jointly transferred, ery-r2-r8/+ genomes are produced in which the r2 marker is stably integrated but the r8 marker is unstably adjoined to its wild-type homolog. Thus, the duplicated region can be quite short. When the ery-r8 marker is stably integrated, the region of the marker is refractory to subsequent transformation. Markers with properties like ery-r8 are not particularly rare, being found with a frequency of about 4% among spontaneous mutations to erythromycin resistance.

Intracellular competition for a mismatch recognition system and marker-specific rescue of transforming DNA from inactivation by ultraviolet irradiation

We show that donor-recipient heterozygosity for unlinked genes allows low efficiency markers in transforming DNA to escape from attack by the discrimination system present in Diplococcus pneumoniae of genotype hex+. The observed efficiency of a marker is a function of donor DNA concentration, duration of exposure, and relatedness of the donor to the recipient strain. Efficiency can be increased at least six to seven fold by addition of excess homologous but nonisogenic DNA, which appears to compete for a limited amount of an enzyme system recognizing mismatched heteroduplexes. Low efficiency markers are often hypersensitive to ultraviolet irradiation of the donor DNA, and this effect also is reversed by the presence of excess nonisogenic homologous DNA, leading to a dramatic (for example, 260-fold) marker-specific rescue from inactivation.

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.

Marker discrimination and mutagen-induced alterations in pneumococcal transformation.

Nitrous acid-induced and hydroxylamine-induced chemical alterations in transforming DNA result in the loss of biological activity and in mutagenesis. The function responsible for the discrimination between high efficiency and low efficiency markers in pneumococcal transformation, and for the elimination of a substantial fraction of spontaneously occurring mutational events, does not appear to act on integrated DNA carrying these chemical alterations. The chemical modifications result in mutations that are evident among bacteria transformed with the treated DNA. Fusidic acid-resistant mutants isolated in this way have been shown to be predominantly of the low efficiency class. We have previously reported that most mutations of spontaneous origin occurring in this locus are of the high efficiency class, and have suggested that discrimination results from the elimination of specific classes of base pair mismatches that occur as intermediates in transformation. It would appear that the base pair mismatches most effectively eliminated in the discriminating strain of pneumococcus are those of the A:C and G:T type and that the immediate product of transformation with mutagenized DNA involves intermediates that are not recognized as A:C or G:T by the discrimination system.

Relation of macromolecular synthesis in streptococci to efficiency of transformation by markers of homospecific and heterospecific origin.

In previous studies with Streptococcus sanguis and S. pneumoniae as recipients and donors of transforming deoxyribonucleic acid (DNA), it was found that heating recipients just prior to exposure to DNA caused an increase in the number of transformants induced by heterospecific DNA relative to that induced by homospecific DNA. In the present studies, S. sanguis recipients were found to recover from this effect of heat (48 C, 15 min) when incubated at 37 C before exposure to DNA. Inhibitors of nucleic acid synthesis, such as rifampin, 5-fluorodeoxyuridine, actinomycin, and p-hydroxyphenylazo-uracil, but not inhibitors of protein synthesis, such as chloramphenicol and erythromycin, prevented recovery from the effect of heat. Inhibitors of nucleic acid synthesis caused changes in unheated cells similar to those observed with heat treatment; these changes included increased transformability by genetically hybrid DNA and by low-efficiency markers in homospecific DNA. The effect of a combination of heat and inhibitors on transformation by heterospecific DNA was greater than when single treatments were used. The most effective inhibitor used alone was rifampin: in treated recipient cells, the yield of transformants produced by a given amount of irreversibly bound heterospecific DNA was increased without a significant change in the yield of transformants produced by bound homospecific DNA. A cell being doubly transformed by homospecific and heterospecific DNA was enhanced specifically in its transformability with the latter as a consequence of rifampin treatment. Treatment with rifampin also increased co-transformation by linked heterospecific markers. The period during which recipient cells were sensitive to the effects induced by rifampin and fluorodeoxyuridine lasted from 10 to 20 min after DNA uptake.

Marker discrimination in transformation and mutation of pneumococcus.

Earlier investigations of pneumococcal transformation revealed a function (hex(+)) responsible for severely reducing the transformation yield of certain markers. A mutational alteration (hex(-)) responsible for the loss of this function has been transferred into a hex(+) strain to permit a comparison of the hex(+) and hex(-) phenotypes in an isogenic background. The loss of the hex(+) function results both in a loss of the capacity to eliminate the low efficiency markers in transformation and a substantial increase in the spontaneous mutation rate. These properties of the hex(-) strain could result from the loss of a capacity to eliminate certain classes of mismatched base pairs that occur as intermediates in both transformation and mutagenesis.

The transformation of both recipient strands of pneumococcal DNA.

SUMMARY: It has been established that, in pneumococcus, donor sites on transforming deoxyribonucleic acid (DNA) can be divided into at least two types, known as low-efficiency (LE) sites and high-efficiency (HE) sites, by comparing the number of transformants of a particular character with that obtained for a standard reference gene ( Ephrussi- Taylor, Sicard & Kamen, 1965 ; Lacks, 1966 ). The reference gene used by Ephrussi- Taylor and her colleagues was str-41, whilst Lacks used the sulf-d gene. Ephrussi- Taylor & Gray (1966) proposed that LE markers are integrated into both chains of the recipient DNA by an excision and repair mechanism, whilst HE markers have to wait for a further division before both the recipient strands become altered. Evidence that the LE markers are transmitted into both strands whilst HE markers are transmitted to only one strand has been put forward by Ephrussi-Taylor (1966) , Gray & Ephrussi-Taylor (1967) and Louarn & Sicard (1968) . Louarn & Sicard (1969) have taken advantage of this difference between the integration of the markers to show that either of the recipient strands of DNA can be transformed by the HE marker. They calculated the ratio R = (D.N)/(A.B), where D = number of doubles transformed for genes A and B, N = the total colony-forming units (c.f.u.) present, and A and B the number of transformants for genes A and B respectively. By comparing the ratios obtained for pairs of markers having the composition HE-HE, LE-LE, or HE-LE, Louarn and Sicard showed that the results were consistent with either strand being transformed by an HE marker. They also discussed the effect of replication events on the value of R, particularly with respect to the location on the chromosome of such events in relation to the markers. The results reported below support these conclusions.

Identical initial steps during transformation for high and low efficiency markers in [formula omitted

Identical initial steps during transformation for high and low efficiency markers in [formula omitted