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We review and extend data showing concerted evolution of parental 18-5.8-26S nuclear ribosomal DNA (18-26S rDNA) gene families in three natural Nicotiana allotetraploids (N. tabacum, N. rustica and N. arentsii, each 2n = 4x = 48) and one synthetic N. tabacum line (Th37, ♀ N. sylvestris (2n = 24) x ♂ N. tomentosiformis (2n = 24)). The origin of the gene families was analysed by sequence polymorphisms in the intergenic spacer (IGS) region and the number of chromosomal loci by fluorescence in situ hybridization (FISH). FISH revealed that the number and locations of 18-26S rDNA in the natural allopolyploids was the sum of those found in the diploid progenitors. However, the rDNA restriction patterns showed polymorphisms in the IGS that were not additive, suggesting that parental rDNA clusters were partially (N. tabacum, N rustica) or completely (N. arentsii) overwritten by hybrid-specific units. Thus the Nicotiana allotetraploids show evidence of concerted evolution, including both intralocus and interlocus gene conversion. A feral N. tabacum collected in Bolivia had a higher proportion of unconverted parental rDNA units than cultivated tobacco varieties, suggesting either that rDNA homogenization is accelerated by inbreeding or multiple origins of tobacco. There is no evidence for the elimination of N. sylvestris-derived rDNA units in the synthetic Th37 tobacco line as occurred in natural tobacco, although several novel rDNA unit variants were found in most but not all the hybrid plants. Factors that may control the occurrence and extent of rDNA homogenization are discussed for allopolyploids in Nicotiana and other taxa.

An ∼135-bp sequence called the A1/A2 repeat was isolated from the transcribed region of the 26-18S rDNA intergenic spacer (IGS) of Nicotiana tomentosiformis. Fluorescence in situ hybridization (FISH) and Southern blot analysis revealed its occurrence as an independent satellite (termed an A1/A2 satellite) outside of rDNA loci in species of Nicotiana section Tomentosae. The chromosomal location, patterns of genomic dispersion, and copy numbers of its tandemly arranged units varied between the species. In more distantly related Nicotiana species the A1/A2 repeats were found only at the nucleolar organizer regions (NOR). There was a trend toward the elimination of the A1/A2 satellite in N. tabacum (tobacco), an allotetraploid with parents closely related to the diploids N. sylvestris and N. tomentosiformis. This process may have already commenced in an S3 generation of synthetic tobacco. Cytosine residues in the IGS were significantly hypomethylated compared with the A1/A2 satellite. There was no clear separation between the IGS and satellite fractions in sequence analysis of individual clones and we found no evidence for CG suppression. Taken together the data indicate a dynamic nature of the A1/A2 repeats in Nicotiana genomes, with evidence for recurrent integration, copy number expansions, and contractions.

Unidirectional gene conversion of rDNA units has occurred in the evolution of natural tobacco (Nicotiana tabacum). In this paper we report the use of the synthetic tobacco line Th37, 4n (N. sylvestris × N. tomentosiformis), to study early rDNA evolution associated with allopolyploidy. At least three classes of newly amplified rDNA unit variants were identified (17/20 plants). Their presence was often accompanied by near-complete elimination of N. tomentosiformis-donated rDNA units (15/20 plants). Novel rDNA units were of N. tomentosiformis-type and contained rearranged subrepeats in the intergenic spacer. The maternal N. sylvestris-derived units were unchanged, except for some alteration in the ratio of individual gene family members. A cytogenetic analysis revealed rDNA sites on N. sylvestris-derived chromosomes S10, S11, and S12 and N. tomentosiformis-derived chromosomes T3 and in some cases T4. An rDNA locus does not occur on N. tomentosiformis chromosome 4. The locus on chromosome T4 of some hybrids correlates with the occurrence of the novel units that probably amplified at the locus. Combined with an analysis of tobacco cultivars, the data indicate that an initial burst of rDNA evolution associated with allopolyploidy was followed by a slower process that led towards reduced complexity and a decreased number of rDNA variants.

We followed the mitotic transmission of an experimentally induced hypomethylated state of several tobacco repetitive sequences in callus culture and plants. The initial hypomethylation was induced by a hypomethylation drug, dihydroxypropyladenine (DHPA), the competitive inhibitor of cellular S-adenosylhomocysteine hydrolase, which is known to preferentially inhibit methylation at CNG and non-symmetrical motifs while having a negligible effect on methylation at CG motifs. The deprivation of this drug resulted in an almost immediate remethylation of cytosines at CNG motifs ( MspI and EcoRII sites) leading us to conclude that, the hypomethylation effect of dihydroxypropyladenine is rather transient and differs from that of 5-azacytidine which often induces heritable changes in methylation patterns. The results suggest that de novo methylation of CNG motifs is a rapid and meiotically independent process on DNA sequences with pre-existing CG methylation.

Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5′-mCmCG-3′ methylation varied significantly between different repeats. There were almost saturation levels of 5-methylcytosine at the inner (3′) cytosine position and variable degrees of methylation at the outer (5′) cytosine at the enzyme recognition sites. The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS. The results suggest that hypomethylation of 5′-mCmCG-3′ sites with DHPA is a gradual non-random process proceeding in the direction mCmCG→CmCG→CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA. Variable decondensation patterns within and between loci were also observed for GRS and HRS60. Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure.

Plants have to cope with a number of envi-ronmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/HpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress.

In order to investigate possible interactions between parental genomes in the composite genome of Nicotiana tabacum we have analyzed the organization of telomeric (TTTAGGG)n and ribosomal gene (rDNA) repeats in the progenitor genomes Nicotiana sylvestris and Nicotiana tomentosiformis or Nicotiana otophora. Telomeric arrays in the Nicotiana species tested are heterogeneous in length ranging from 20 to 200 kb in N. sylvestris, from 20 to 50 kb in N. tomentosiformis, from 15 to 100kb in N. otophora, and from 40 to 160kb in N. tabacum. The patterns of rDNA repeats (18S, 5.8S, 25S RNA) appeared to be highly homogeneous and speciesspecific; no parental rDNA units corresponding to N. sylvestris, N. tomentosiformis or N. otophora were found in the genome of N. tabacum by Southern hybridization. The results provide evidence for a species-specific evolution of telomeric and ribosomal repeats in the tobacco composite genome.

Previously, we established experimental conditions allowing us to induce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezděk et al. 1991, Planta 184, 487–490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-differentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 105 copies in the genome, can escape de-novo methylation.

Several closely related members of HRS60 family of repeated DNA sequences of Nicotiana tabacum genome were recently cloned. In this paper a relative amount of HRS60-like DNA repeats was established in 19 Nicotiana species using slot-blot DNA/DNA hybridization with HRS60.1 repeat under three different stringencies. At high stringency the strongest hybridization signal gave DNAs of N. sylvestris and N. tabacum, the former species being one of the progenitors of N. tabacum. Hybridization with other Nicotiana species gave signals the strength of which did not exceeded 3% of that of N. sylvestris. This indicates that HRS60.1 repeat and its close relatives were specifically amplied in N. sylvestris and preserved in the evolution of N. tabacum allotetraploid genome without substantial changes. The relative abundance of these repeated DNA sequences enables to use them as a useful molecular genetic marker. At lower stringencies of DNA/DNA hybridization elevated levels of relative hybridization signals in several species were observed. This indicates that also DNA repeats related to HRS60.1 are present in the genus Nicotiana.

Genomic instability of protoplast-derived tobacco plants was studied by means of phenotypic evaluation, karyological analysis, and Southern blot experiments. Of the total number of 91 regenerants belonging to 35 different protoclones 57 plants displayed various morphological and/or functional aberrations, some of them being inherited into the progeny. A karyological study of 20 randomly chosen plants revealed 15 tetraploid and 5 diploid chromosome sets. A Southern blot hybridization analysis of three regenerants displayed some DNA polymorphism (RFLP) and thus confirmed that in such plants alterations in the genome structure could be found and that genotypes of protoplast-derived plants frequently differ from the parental genotype.

In pentaploid dogroses, Rosa section Caninae (2n=5x=35), the pollen transmits one basic genome (x=7) derived from the seven segregating bivalents, whereas the egg transmits four basic genomes (4x=28) one set derived from the segregation of seven bivalents and three sets of univalent-forming chromosomes. Chromosomes from all five genomes carry 18-5.8-26S nuclear ribosomal DNA (rDNA) sites. This mode of sexual reproduction, known as permanent odd polyploidy, can potentially lead to the independent evolution of rDNA on bivalent- and univalent-forming chromosomes. To test this hypothesis, we analyzed rRNA gene families in pollen and somatic leaf tissue of R. canina, R. rubiginosa and R. dumalis. Six major rRNA gene families (alpha, beta, beta' gamma, delta and epsilon) were identified based on several highly polymorphic sites in the internal transcribed spacers (ITSs). At least two of the major rRNA gene families were found in each species indicating that rDNAs have not been homogenized across subgenomes. A comparison of ITS1 sequences from leaf and pollen showed differences: the shared beta rRNA gene family was more abundant among pollen clones compared to leaf clones and must constitute a major part of the rDNA loci on bivalent-forming chromosomes. The gamma and delta families were underrepresented in pollen genomes and are probably located predominantly (or solely) on the univalents. The results support the hypothesis that pentaploid dogroses inherited a bivalent-forming genome from a common proto-canina ancestor, a likely donor of the beta rDNA family. Allopolyploidy with distantly related species is likely to have driven evolution of Rosa section Caninae.

ABamHI family of highly repeated DNA sequences of theNicotiana tabacum nuclear genome, denoted as a HRS60-family, was recently isolated. It comprises about 2% of the tobacco nuclear genome. Monomeric units are 182–184 bp long. Members of the HRS60-family isolated till now are closely related. DNA-DNA hybridization experiments with DNA of the two tobacco progenitors,N. tomentosiformis andN. sylvestris, revealed that the HRS60-family was present in many copies inN. sylvestris, the amount being about 1.7 times that inN. tabacum. InN. tomentosiformis as well as in some other species of the genusNicotiana, the HRS60-family is present in a small amount. Sequences related to the HRS60-family were revealed using DNA-DNA hybridization at low stringency. With respect to quantity, the HRS60-family could be considered as a species-specific DNA repeat which may be a useful genetic marker in genetic manipulations withN. tabacum.

Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5'-mCmCG-3' methylation varied significantly between different repeats. There were almost saturation levels of 5-methylcytosine at the inner (3') cytosine position and variable degrees of methylation at the outer (5') cytosine at the enzyme recognition sites. The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS. The results suggest that hypomethylation of 5'-mCmCG-3' sites with DHPA is a gradual non-random process proceeding in the direction mCmCG--CmCG--CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA. Variable decondensation patterns within and between loci were also observed for GRS and HRS60. Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure.

We have recently shown that hypomethylation of cytosine residues in the HRS60 family of repetitive DNA sequences can be induced with 5-azacytidine (5-azaC) in tobacco tissue cultures. We have also proven that such a DNA methylation status is maintained during the recovery of protoplasts, plant regeneration, and vegetative development. In the present paper we follow meiotic transmission of hypomethylated HRS60 DNA. Plants obtained from seeds treated with 5-azaC were either self pollinated or crossed with a non-treated control in a reciprocal way. Analysis of the methylation status of the HRS60 DNA revealed that these sequences were hypomethylated in the progenies up to the extent found in the parental 5-azaC-treated plant. Since no parent-of-origin effect was observed, we presume that both male and female gametes transmit an artificial methylation imprint to a similar extent. This result is supported by methylcytosine evaluation in the total genomic DNA samples. A temporal analysis of 5-azaC effects on germinating seeds and a phenotypic evaluation of 5-azaC-treated tobacco plants are also presented.

Higher plant DNA is methylated at CG and CNG targets. In this study we have investigated the tobacco methylation system in tissue culture using the methylation inhibitors 5-azacytidine (5-azaC), dihydroxypropyladenine (DHPA) and ethionine (Ethi), and methylation-sensitive restriction endonucleases HpaII, MspI, HhaI, EcoRII, ScrFI, and Fnu4HI. Surprisingly, CAG/CTG sequences, contrary to CG doublets and CCG/CGG triplets, appeared to be refractory to the inhibitory effect of 5-azaC. Thus 5-azaC cannot be considered a general inhibitor of DNA methylation in tobacco cells. On the other hand, DHPA, the inhibitor of S-adenosylhomocysteine (SAH) hydrolase, and Ethi caused hypomethylation of both CAG/CTG and CCG/CGG triplets but not of the CG doublets. The sensitivity of triplet-specific methylation to the inhibition of SAH hydrolase suggests the possibility that plant-specific DNA methylation at CNG targets might be modulated by alterations of the SAH/S-Adenosyhnethionine ratio in plant cells.

Of the cells of Lactobacillus acidophilus R-26 incorporated 3H-thymine (specific radioactivity 1.57 Ci/mmol or 3.15 Ci/mmol), their transfer to a medium without essential amino acids resulted in their death. This death may be interpreted in such a way that cell damage caused by disintegration of tritium cannot be effectively repaired under conditions of amino acid deprivation. The experimental eonditions make it possible to explain this death either as a result of inhibition of protein or RNA synthesis or as a result of the absence of amino acids. These possibilities were tested in experiments, in which the synthesis of proteins and RNA was inhibited by specific inhibitors in the presence of amino acids. Under these conditions no death of cells was detected, thus indicating that free amino acids play a role in the repair of radiation damage.

A study was made of the SOS induction of the gene sulA of Escherichia coli K12 in relation to the gene dosage of the gene recA. In experiments the sulA::lacZ fusion strain PQ37 and derivatives of PQ37 with the multi-copy plasmids pDR1453 or pBR322 were used. The SOS response was induced with nitrofurantoin, SOS induction of the gene sulA was determined on the basis of the amount of beta-galactosidase synthesized, i.e. by the SOS chromotest (Quillardet et al., 1982a). It was found in this work that cells with the plasmid pDR1453, which contain the gene recA of E. coli K12 (Sancar and Rupp, 1979), have a decreased SOS induction of the gene sulA. Cells with the plasmid pBR322 do not exhibit this decrease. Inactivation of the gene recA in the plasmid pDR1453 with preservation of the functional gene recA in the chromosome leads to a restoration of 'standard' SOS induction of the gene sulA. The results show that the amount of the gene product of the gene recA affects the SOS induction of the gene sulA.

Several simple furan and nitrofuran compounds were tested for mutagenicity and related biological activities, in the Ames test, the bacterial repair test, the prophage-induction test and the chloroplast-bleaching test. Only those compounds having the nitro-group were found to be active in the test. If the nitro-group was in the β instead of in the α position, the mutagenic activity was much reduced (0.3 revertants per nanomole, the other simple nitrofurans producing 5–15 reversions per nanomole, as did 5-nitrofuran and 5,2-dinitrofuran). The vinyl-group-containing compound, 5-nitro-2-furylacrylic acid, was by one decadic order more effective both in the Ames test and in the prophage-induction test. In the repair tests all nitrofurans displayed a much higher dependence on the recA-repair system than on the uvrA system. For 2 compounds, the dinitrofuran and, especially, the 5-nitrofuraldehyde, the uvrA cells were even less sensitive than the wild-type cells.

Induced mutagenesis was studied in Escherichia coli K12 cells in relation to the level of RecA-protein (P-RecA). In experiments strains AB2497, AB2497(pBR322) and AB2497(pX02) were used. The multicopy plasmid pX02 is a recombinant of pBR322 and recA+ gene of E. coli K12. Cells carrying this plasmid overproduce the P-RecA constitutively. Mutagenesis was induced by the decay of incorporated 6-3H-thymidine. Mutations of the argE3 (ochre) to Arg+ prototrophy were followed. Besides the frequency of mutations, mutagenic specificity was determined. In cells AB2497(pX02) which overproduce the P-RecA the yield of Arg+ revertants was markedly reduced compared with that in strains AB2497 or AB2497(pBR322), whereas the mutagenic specificity was not changed. In all the strains studied the predominant type of mutation produced was the base substitution in the A:T base pair.

HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved "relic" DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a "HRS60-family". Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.

In E. coli cells with potentially lethal damage to their DNA are starved of amino acids they rapidly lose viability (Koukalová and Kuhrová 1980). This phenomenon is called secondary lethality (SL). In the course of secondary lethality there is a decrease in the molecular weight of DNA to a value on average three times lower (DNA fragmentation, and part of the DNA is reduced to acid-soluble fractions (DNA degradation). Caffeine inhibits SL, and both these processes of DNA decay, The lowest effective concentration 5 mg/ml, with maximum effect at a concentration of 10 mg/ml. SL is also inhibited by the absence of an energy source. The possible mechanism of SL is discussed on the basis of these results.

Unlike in most bacterial species, so far studied synthesis of DNA inLactobacillus acidophilus R-26 continues even in the absence of protein synthesis or in the absence of essential amino acids, the increase of DNA under these conditions being 100–150%. These increases not only correspond to termination of running replication cycles of DNA but also to onset and termination of additional new cycles. In the absence of amino acids this “relaxed” DNA synthesis (as well as RNA synthesis) is stimulated by deoxyadenylate and chloramphenicol. On the other hand, it is inhibited by actinomycin D or rifampicin. In the presence of these inhibitors of RNA synthesis the increase of DNA does not exceed 40%, even when compounds stimulating DNA synthesis,i.e. deoxyadenylate or chloramphenicol are simultaneously present. However, when free amino acids are present, either all or only glutamic acid and aspartic acid, the “relaxed” DNA synthesis takes place too even if the synthesis of proteins and RNA is simultaneously inhibited. It is assumed that transcription of RNA, and also free amino acids or their non-proteinic derivatives play a significant role in control of the chromosomal replication cycle.

Growth ofLactobacillus acidophilus was inhibited in the presence of deoxyadenosine-5′-monophosphate (dAMP). The other purine deoxyribonucleotides and -sides were only weak inhibitors, and pyrimidine deoxyribotides and -sides were inactive. dAMP did not act as an inhibitor if thymine, thymidine, or 5-methyldeoxycytidine-5′-monophosphate was present in the medium. The inhibition by dAMP was counteracted by increasing concentrations of deoxyuridine, deoxyuridine-5′-monophosphate and, to some extent, adenosine-5′-monophosphate. The effect of these substances was proportional to their concentration and competitive in character. The results support the assumption that dAMP inhibits the synthesis of thymine. Mutants ofL. acidophilus resistant to inhibition by dAMP were found.

In Lactobacillus acidophilus , the DNA synthesis in cells incubated in the absence of essential amino acids reaches levels corresponding to the initiation of further replication cycles than just to completing the cycles already running. This “relaxation of DNA synthesis” is stimulated by the presence of inhibitors of protein synthesis and by the presence of deoxyadenylate. These enhacements of DNA replication are cancelled by actinomycin D. In the presence of inhibitors of protein synthesis, the relaxation of DNA synthesis is further stimulated by free amino acids. The effect of free amino acids, unlike in the former cases, is not inhibited by actinomycin D. It is proposed that the chromosome replication in Lactobacillus acidophilus is regulated similarly as in some plasmids, i.e., independently of the synthesis of protein, depending on the synthesis of RNA and, in addition, on the presence of free amino acids.

Solutions of aflatoxins B1, B2, G1 and G2 and solid films of aflatoxin B1 were irradiated with UV light of wavelength corresponding to the absorption band of aflatoxins at approximately 365 nm (approximately 27 500 cm-1). The reaction was followed spectrophotometrically. Simultaneously the mutagenicity of samples of aflatoxins was determined using Salmonella typhimurium/mammalian microsome test. UV-irradiation caused a modification in all aflatoxins studied (in aqueous and nonaqueous solutions as well as in a solid film); the final photoproducts lost completely the mutagenic activity.

Cells containing incorporated 3H-thymidine are damaged by its decay. It was found with E. coli TAU-bar cells that a small part of the damage is lethal whereas most of it is reparable and only potentially lethal. If cells are subjected to nongrowth conditions, the potentially lethal damage changes to lethal damage. This process is called secondary lethality (SL). The extent of SL and some changes in DNA under three different modes of growth inhibition were determined. It was found that: (i) SL is maximal under conditions of amino acid starvation (-AA), the viable count decreasing by two orders of magnitude. (ii) SL is 4 times lower in the presence of chloramphenicol (-AA + CLP) and 6.5 times lower under + AA + CLP conditions. Changes in the sedimentation rate of DNA determined in alkaline sucrose gradient correlate with the differences in SL: under -AA conditions the sedimentation rate of DNA decreases whereas in the presence of CLP no decrease occurs. The results suggest that certain enzymatic processes take place under -AA conditions which lead to irreparable changes in DNA.

A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions. The host cells used were E. coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir). An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that the recA protein protects phage DNA during the process of restriction. A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth. By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.

Chloramphenicol (CLP) at slightly inhibitory concentrations (3-5 microgram/ml) suppresses the inhibition of DNA synthesis and cell division caused by the temperature sensitive mutation dnaE486 at the nonpermissive temperature. Some other mutations can also be phenotypically suppressed by CLP. A similar effect is shown by chlortetracycline. Phenotypic suppression is caused by both these drugs in different cases than by streptomycin.

Replication of the bacterial chromosome was studied in two substrains ofLactobacillus acidophilus R-26 during amino acid starvation. According to the hypothesis of Maaloe and Hanawalt (1961), already initiated DNA replication cycles are completed under such conditions, with a corresponding 40% increase in the DNA content; new cycles cannot be initiated in the absence of proteosynthesis. Our findings are considerably at variance with this hypothesis. It was found that the course of DNA synthesis and the size of DNA increments during amino acid starvation were influenced by some low molecular weight substances, in particular by deoxyadenylate and spermidine. In the presence of these substances in media without the essential amino acids, prolonged DNA synthesis accompanied by large DNA increments was observed, suggesting that new DNA replication cycles were initiated. The possibility that deoxyadenylate and spermidine influence the regulation of synthesis of the bacterial chromosome is discussed.

Among other temperature-sensitive mutants ofLactobacillus acidophilus the mutant “ts 9” with temperature-sensitive initiation of DNA synthesis was isolated. In this mutant, the course of DNA synthesis under non-permissive conditions proceeds in two phases. During the first 90–120 min, a slight increase (20–50%) of DNA content takes place. Then during further incubation at 40°C, the capacity for initiation of further DNA synthesis increases and a second round of DNA synthesis starts after 3–4h of incubation. The initiation of DNA synthesis is prevented by chloramphenicol and the preceding lag is temperature-dependent. It is concluded that an accumulation of an initiation factor is required for the onset of a new cycle of DNA synthesis and that in thets 9 mutant this accumulation is inhibited at non-permissive temperature.

The synthesis of DNA was investigated inLactobacillus acidophilus R-26 during starvation for glutamic acid. The synthesis of RNA and of proteins was also examined. The content of DNA in the cell increased by 150–250%. The results show that new DNA-replication cycles can be initiated without simultaneous synthesis of proteins and of RNA.

The amino acid regulation of RNA synthesis inLactobacillus acidophilus was studied and found to be of stringent character. The synthesis of RNA was inhibited in the absence of essential amino acids in the medium, this inhibition being released by chloramphenicol or chlortetracycline. The RNA synthesized in the presence of the above inhibitors was not stable. The results do not support the hypothesis that the release of RNA synthesis by chloramphenicol is due to an increased pool of free amino acids, in consequence to the inhibition of protein synthesis. Chloramphenicol removed the inhibition of RNA synthesis at the same rate as the amino acids themselves. The pool of free leucine or histidine decreased to 60% in the absence of exogenous amino acids and it was not raised on adding chloramphenicol. The results are in agreement with the assumption that the synthesis of ribosomal RNA in bacteria is controlled by the equilibrium between polysomes and free ribosomes. Further, the results point to a possible limiting role of proteins in the regulation of ribosomal RNA synthesis.

Injected stem cells of the bone-marrow of mice form colonies of haematopoietic tissue in the spleen of an irradiated host and this can be used to obtain stem-cell survival curves after irradiation in vivo or in vitro (Till and McCulloch 1961). The repopulation capacity of bone-marrow cells after fractionated irradiation has been studied, too (Porteous and Lajtha 1966). This paper reports changes in the count of stem cells in the bone-marrow of mice after continuous irradiation by gamma-rays at a low dose-rate. Both male and female C57B mice were used; at the start of irradiation they were 12 weeks old. The irradiation was applied continuously for 24 hours a day and was interrupted every other day for about 10 min to replenish food and clean the cages. The animals were placed into the irradiated space at various intervals so as to obtain groups given accumulated doses of from 40 to several hundred R. All animals used for a given series of experiments stemmed from the same group of the same age. For each series the stem-cell counts of irradiated mice were compared with non-irradiated controls. These controls were kept throughout the irradiation in the same space, but their exposure was prevented by concrete shields of proper dimensions. Thus the controls could not receive doses larger than 2 mR/day. So far, three different dose-rates have been tested-24-0 R, 8-0 R and 27 R per 24 hours. The mice were killed after the irradiation was completed, and a suspension of cells in Hanks' solution was prepared from the bone-marrow of tibiae and femurs. This suspension was injected into host animals of the same strain in doses of 105 nucleated elements per mouse. Host mice were irradiated by a dose of 650 R twenty hours before injection. By the 10th day after injection the mice were killed, and colonies of the haematopoietic tissue at the facies parietalis of the spleen were counted. The individual points in the plots are the mean values estimated for groups consisting of 12-15 mice. The background value, 0-3-0-6 colony per mouse, was subtracted. The results show that the count of stem cells in the bone-marrow responds to continuous irradiation by a decrease at all three dose-rates (figure). The initial decrease is the same at 24 R and 8 R per day, whereas a somewhat lower decrease was brought about by the same dose accumulated at a daily rate of 2-7 R, the difference, however, being not statistically significant. Accumulated doses larger than 200 R were tested only at daily rates of 24 R and 2-7 R. The difference between both plots is considerable. With several groups there has been observed at 27 R/day a recovery to normal values followed by another decrease, and this course recurred. As this experiment is rather timeconsuming at a rate of 27 R/day, it was carried out only twice. The same results could be obtained also for the second run, i.e. an alternating decrease to R.B.