B. Koukalová

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

SummaryHRS60.1, 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.

Isolation and characterization of two middle repetitive DNA sequences of nuclear tobacco genome.

SummaryTwo DNA sequences, R8.1 and R8.3, representing two distinct classes of tobacco genomic repeated DNA, were cloned and characterized by Southern blot analysis. Both R8.1 and R8.3 were found to be homologous to the Nicotiana tomentosiformis component of the allotetraploid Nicotiana tabacum genome, and each of them represents about 0.3% of nuclear DNA. The R8.1 and R8.3 differ in the mode of distribution in chromosomes, as revealed by in situ DNA/DNA hybridization.

The distribution of tobacco HRS60 DNA repeated sequences in species of the genus Nicotiana

Abstract 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.

Mutagenic activities of simple nitrofuran derivatives. I. Comparison of related compounds in the phage inductest, chloroplast-bleaching and bacterial-repair and mutagenicity tests.

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 beta instead of the alpha 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 urA cells were even less sensitive than the wild-type cells.

Genome modifications in protoplast-derived tobacco plants: Phenotypic evaluation and RFLP Analysis

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.

Inhibition of thymine synthesis inLactobacillus acidophilus R-26 by deoxyadenosine-5′-monophosphate

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.

Protection of nonmodified phageλ againstEcoK restriction mediated byrecA protein

A study was conducted to establish whether theEcoK-specific restriction, which is alleviated inE. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level ofrecA protein without induction of other SOS functions. The host cells used wereE. coli K-12 ,strain AB2497, and its derivatives; the nonmodified phage λ was a mutant b2b5(vir). An increase of therecA protein level was induced using the plasmid pX.02, which is a recombinant of pBR322 carrying therecA gone ofE. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that therecA 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.

Stringent control of RNA synthesis inLactobacillus acidophilus

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.

ABam HI family of tobacco highly repeated DNA

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.

A mutant ofLactobacillus acidophilus with temperature-sensitive regulation of DNA synthesis

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.