Sequence analysis of Vicia faba highly repeated DNA: the BamHI repeated sequence families

Cleavage of Vicia faba nuclear DNA with the restriction endonuclease BamHI yielded discrete size classes of 250, 850, 900, 990, 1 150, 1 500 and 1 750 bp of highly repetitive DNA. Each of these sequence families comprised about 3% of the total genomic DNA. Some sequence members from each sequence family were cloned in pBR322 and their primary structures determined. Computer analyses of nucleotide sequences suggested the existence of about 60 bp sequence periodicity within the repeating unit of the 990 bp sequence family, though the extent of homology among the surmised shorter subrepeat units was very low. With other BamHI sequence families, however, the data did not show any clear internal sequence periodicity. The repeat units of the 850 bp and 1 750 bp sequence families contained nucleotide sequences homologous to the 250 bp family sequence. No sequence relationship between or among other sequence families was observed. There was 13–25% sequence variation among 6 cloned members of the 250 bp family and probably also among those of other BamHI repeat families. DNA sequences homologous to these V. faba BamHI repeat families were detected in Pisum sativum DNA by Southern blot hybridization. Furthermore, very weak cross-hybridization was observed with plant DNAs from Phaseolus vulgaris, Triticum aestivum, Cucumis sativus and Trillium kamtschaticum.     

Effect of repeated DNA sequences on direct gene transfer in protoplasts of Nicotiana plumbaginifolia

Summary Highly repeated nuclear DNA sequences from leaves of Nicotiana plumbaginifolia were cloned in pBR322 and tested for their effect on direct gene transfer in protoplasts of the same organism. Protoplasts were prepared from suspension cultures and were incubated in the presence of the plasmid pHP23 carrying the kanamycin resistance gene APH(3)II and in the presence of the plasmids carrying the cloned sequence. DNA uptake was induced by a polyethyleneglycol (PEG) treatment. Out of the 22 tested clones, 3 significantly stimulated the frequency of appearance of transformed colonies. DNA was extracted from some of the kanamycin-resistant calli obtained by co-transformations. Dot-blots have shown that the stimulatory effect on transformation frequency is often accompanied by a consistent increase in integrated genes sequences.     

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.     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.     

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

Summary Two 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.     

Variation in nuclear DNA content of isonicotinic acid hydrazide-resistant cell lines and mutant plants of Nicotiana tabacum

Summary Isonicotinic acid hydrazide (INH)-resistant lines of Nicotiana tabacum have been maintained in callus culture for six years and mutant plants have been regenerated from a number of these lines. This study examines variations in DNA content in nuclei of several of these callus cultures, regenerated plants, and secondary callus from the regenerated plants. The lines selected for study include three easily regenerated lines (I 21, I 24, and I 9) and two lines of poor regenerating capacity (I 1 and I 18). Two of the regenerating lines eventually led to fertile plants and the third produced only sterile plants. In general, the range of total nuclear variability was not as high as anticipated from other studies of long-term tobacco callus cultures. The majority of nuclei in all the distributions were between 3 and 20 pg, and the most frequently encountered distributions concentrated in the 7–18 pg region corresponding to 2–5C by our estimate of the C value for tobacco. Distributions were not identical for plants regenerated from the same culture simultaneously, and the nuclear DNA content of secondary callus cultures from one of the plants examined did not reflect the quantitative DNA pattern of the plant from which it was derived. The greatest degree of variability and highest DNA content for individual nuclei were observed in the primary callus of the poorly- and non-regenerating lines. The variability in DNA content was not associated with the INH-resistant trait.     

Further characterization of picloram-tolerant mutants of Nicotiana tabacum

Summary A genetic and preliminary biochemical analysis has been performed on four picloram-tolerant mutants of Nicotiana tabacum that were isolated from cell cultures. The four mutations define three distinct linkage groups. Mutant seedlings incorporate radioactively labeled picloram normally and do not modify or degrade the herbicide in a manner that alters its solubility characteristics.     

Immobilization of cells of Nicotiana tabacum L. on polyphenyleneoxide coated with poly-L-lysine

Cells of Nicotiana tabacum L. cv. Wisconsin 38 were immobilized on poly (2,6-dimethyl)-p-phenyleneoxide in powder form (Sorfix) coated with poly-L-lysine (molecular weight 40 000 daltons). The dependence of cell immobilization on the amount of bound polyL-lysine was estimated.Abbreviations MW molecular weight - dwt dry weight - fwt fresh weight     

Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.     

Is bromodeoxyuridine resistance a consequence of cytokinin habituation in Nicotiana tabacum?

Cytokinin-habituated, as compared to nonhabituated Nicotiana tabacum cv. Havana 425 callus was shown to tolerate higher concentrations of 5-bromodeoxyuridine (BUdR) if cytokinin was added to the medium [Meins, Planta 129, 239–244 (1976)].Our aim was to clarify whether or not BUdR-resistance and cytokinin-habituation are linked characters in callus cultures of our BUdR-resistant tobacco mutant BR 37/21, obtained from Nicotiana tabacum cv. Ottawa.It is demonstrated that BUdR-resistance and cytokinin-habituation are independent characters in the case of this mutant. Non-habituated BR 37/21 callus grows as a resistant line on a medium containing BUdR at a selective concentration (30 mg/l) whereas cytokinin-habituated callus, not selected for BUdR-resistance, behaves as a sensitive line, i.e. turns brown and dies, on the same medium.     

Differential sensitivity of CG and CCG DNA sequences to ethionine-induced hypomethylation of the Nicotiana tabacum genome.

Plant DNA is distinguished from the DNA of all other organisms by its high content of 5-methylcytosine (5mC). 5mC levels may amount to 30% of total cytosines, distributed between the sequences CG and CXG. The results presented here show that the methylation status of CXG sequences could be influenced by culturing tobacco tissues on subtoxic concentrations of ethionine. The hypomethylating effect of ethionine, evaluated as the capability of MspI or HpaII to cleave the DNA, proved to be rather specific for CCG and differed from that or 5-azacytidine which did not discriminate between CG and CXG sequences.     

Differential sensitivity of CG and CCG DNA sequences to ethionine-induced hypomethylation of the Nicotiana tabacum genome

Plant DNA is distinguished from the DNA of all other organisms by its high content of 5-methylcytosine (5mC). 5mC levels may amount to 30% of total cytosines, distributed between the sequences CG and CXG. The results presented here show that the methylation status of CXG sequences could be influenced by culturing tobacco tissues on subtoxic concentrations of ethionine. The hypomethylating effect of ethionine, evaluated as the capability of MspI or HpaII to cleave the DNA, proved to be rather specific for CCG and differed from that or 5-azacytidine which did not discriminate between CG and CXG sequences.     

Production of somatic hybrids between Daucus carota L. and Nicotiana tabacum

Protoplasts of a kanamycin-resistant (KR, nuclear genome), streptomycin-resistant (SR, chloroplast genome) and chlorophyll-deficient (A1, nuclear genome) Nicotiana tabacum (KR-SA) cell suspension cultures or X-ray-irradiated mesophyll protoplasts of kanamycin- and streptomycin-resistant green plants (KR-SR) were fused with protoplasts of a cytoplasmic male-sterile (CMS) Daucus carota L. cell suspension cultures by electrofusion. Somatic hybrid plants were selected for kanamycin resistance and the ability to produce chlorophyll. Most of the regenerated plants had a normal D. carota morphology. Callus induced from these plants possessed 23–32 chromosomes, a number lower than the combined chromosome number (66) of the parents, and were resistant to kanamycin, but they segregated for streptomycin resistance, which indicated that N. tabacum chloroplasts had been eliminated. Genomic DNA from several regenerated plants was analyzed by Southern hybridization for the presence of the neomycin phosphotransferase gene (NPTII); all of the plants analyzed were found to contain this gene. Mitochondrial (mt) DNA was analyzed by Southern hybridization of restriction endonuclease digests of mtDNA with two DNA probes, PKT5 and coxII. The results showed that the two plants analyzed possessed the mitochondria of D. carota. These results demonstrate that the regenerated plants are interfamilial somatic hybrids.     

Genotypic control of pollen plant formation in Nicotiana tabacum L.

Summary Tobacco plants (Nicotiana tabacum L.) of four varieties (Badischer Burley, White Burley, Techne, Kupchunos) were raised at different temperatures and daylengths and the effect of genotype on embryogenic pollen grain formation in situ and on pollen plant formation in anther and pollen cultures from these plants was studied. Genotype controlled embryogenic pollen grain and pollen plant formation by defining productivity under standard growth conditions (long days at 24 °C). Kupchunos was the most productive variety, followed by White Burley, Techne, and Badischer Burley. Furthermore, genotype defined which environmental factor was able to affect embryogenic pollen grain and pollen plant formation and also to which degree. In anther cultures, in addition to these effects, genotype controlled the formation of (an) inhibitory substance(s) in the anther wall in interaction with the plant growth conditions. In Badischer Burley and Techne, inhibitor action could be prevented by isolation of the pollen after one week of anther culture. Finally, direct pollen cultures in Badischer Burley and Techne produced embryos were only when the pollen was isolated from nearly mature anthers, while in White Burley and Kupchunos, embryos also produced at earlier stages and at higher yields. This indicated that genotype controls the time when the embryogenic pollen grains become ready to divide. The results are discussed in relation to strategies to overcome recalcitrance of species and genotypes.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Genetic diversity among sublines originating from a single somatic hybrid cell of Duboisia hopwoodii + Nicotiana tabacum

Summary The genetic instability of an intertribal hybrid cell line, Duboisia hopwoodii + Nicotiana tabacum, obtained by mechanical isolation of a single hybrid cell was studied. Ten subclones of calli derived from this hybrid cell line were cultured for 3 years, and their genetic makeup clarified as to nuclear DNA content, chromosome constitution, and peroxidase isozymes. Nuclear DNA content differed in each subclone. In most subclones, mean DNA content was lower than the mean DNA content in the original hybrid cell line determined 1 year after fusion. This decrease in DNA content is partly attributable to the elimination of tobacco chromosomes that occurred in all subclones. The extent to which tobacco chromosomes were eliminated varied among the subclones — evidence that chromosome elimination occurred slowly. Peroxidase isozyme analysis indicated the loss of a tobacco-specific isozyme, thus confirming results obtained by chromosome analysis. Shoots regenerated from two hybrid subclones after 2 years were also heterogeneous in morphology and nuclear DNA content.     

Zearalenone and flower bud formation in thin-cell layers of Nicotiana tabacum L.

Three lines of evidence indicated a connectionbetween zearalenone (ZEN) and flower bud formationin thin cell layer (TCL) explants of Nicotianatabacum L. cv. Samsun. (1) There were two peaks inthe endogenous ZEN level during the formation offlower buds. (2) The specific inhibitor of ZENbiosynthesis, malathion (MAL), inhibited thebiosynthesis of endogenous ZEN and at the same timeflower bud neoformation. (3) Exogenous ZEN inducedflower bud neoformation.     

Isolation and characterization of isonicotinic acid hydrazide-resistant mutants of Nicotiana tabacum

Summary Twenty stable variant lines resistant to isonicotinic acid hydrazide (INH), an inhibitor of the conversion of glycine to serine in the glycolate pathway, were isolated in cell cultures initiated from allodihaploid Nicotiana tabacum. Plants were regenerated from 13 of these lines and explants were tested for resistance. For some lines virtually all of the regenerated plants scored as resistant; for others a mixed population of sensitive and resistant plants were obtained. One or more plants from 5 lines were fertile, presumably as a result of spontaneous diploidization of cells in the plant or culture. Callus initiated from the seed progeny of these plants was resistant to INH confirming the characteristic as a stable mutation. Seedlings from all INH-resistant plants were small and slow-growing, but the slow-growth trait could be separated from resistance in backcrosses of hybrids. In one case (line I21) crosses with sensitive lines show the resistant trait in that line to be dominant.     

Structure and evolution of a family of interspersed repetitive DNA sequences inCaenorhabditis elegans

Summary The structure of three members of a repetitive DNA family from the genome of the nematodeCaenorhabditis elegans has been studied. The three repetitive elements have a similar unitary structure consisting of two 451-bp sequences in inverted orientation separated by 491 bp, 1.5 kb, and 2.5 kb, respectively. The 491-bp sequence separating the inverted 451-bp sequences of the shortest element is found adjacent to one of the repeats in the other two elements as well. The combination of the three sequences we define as the basic repetitive unit. Comparison of the nucleotide sequences of the three elements has allowed the identification of the one most closely resembling the primordial repetitive element. Additionally, a process of co-evolution is evident that results in the introduction of identical sequence changes into both copies of the inverted sequence within a single unit. Possible mechanisms are discussed for the homogenization of these sequences. A direct test of one possible homogenization mechanism, namely homologous recombination between the inverted sequences accompanied by gene conversion, shows that recombination between the inverted repeats does not occur at high frequency.