Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Characterization of spelt (Triticum spelta L.) forms by gel electrophoretic analyses of seed storage proteins. I. The gliadins.

A comparison betweeen the electropherograms of the spelt and wheat cultivars showed specific differences in the gliadin band patterns which provided the possibility of a clear classification into spelt or wheat. A special nomenclature was developed to be able to improve the presentation of the gliadin band pattern of spelt, which is different from that of wheat. This nomenclature, however, has not yet been applied to other cereals. The gliadin band patterns were presented in a schematic form. As a parameter for comparison, idealized band patterns of both wheat and spelt were developed by comparing the proportions of the bands of all available types. When comparing the gliadin band patterns of the spelt cross-breeds with their corresponding parental generations, it was noted that the same parental bands were not always transmitted and that the cross-breeds showed differences in the intensity, mobility, occurrence, and the splitting of single bands. In general it can be said that the band pattern of the daughter generation - even in the examined F(5) and F(6) generations - is more similar to the band pattern of the mother than to that of the father, which proves a maternal effect.

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants.

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.

Mitochondrial DMA variation in plants regenerated from embryogenic callus cultures of CMS triticale.

The mitochondrial DNA (mtDNA) organization of primary hexaploid cytoplasmic male-sterile (CMS) triticale regenerants containing Triticum timopheevi cytoplasm was analysed by hybridization experiments and compared with the mitochondrial genome organization of the corresponding regenerants with maintainer cytoplasm. Callus cultures had been derived from immature embryos, and 623 triticale plants were regenerated via somatic embryogenesis after three to four subcultures. The chondriome of 159 regenerants was investigated with regard to somaclonal variation. Six different mitochondrial gene probes and four different restriction enzymes were used for Southern blot analyses by the non-radioactive digoxigenin labeling technique. Alloplasmic regenerants showed a gain or loss of hybridization signals up to a high percentage, while euplasmic ones revealed only minor variability with respect to band stoichiometries. In 24 cases rearrangements in the mtDNA were proved. We suppose that recombination processes and selective amplification events are responsible for these findings.

Non-radioactive organization and transcript analysis of the ATPase subunit 6 gene region in the mitochondrial genome from fertile and sterile (CMS) forms of wheat and triticale.

In sterile triticale forms [with cytoplasmic male-sterile (CMS)-inducing timopheevi cytoplasm], fertile orms (with normal cytoplasm) and the corresponding wheat cross parents mitochondrial DNA (mtDNA) and RNA (messenger mtRNA) were characterized using total DNA and RNA material for Southern and Northern blots. A novel non-radioactive technique was applied by marking the probes with digoxenin. The fertile and sterile Triticum and triticale forms were analysed in three genes, atp 6, coxIII and rps13. These forms can be distinguished in the apt6 gene at the mtDNA and mtRNA levels.

Banding of rye (Secale cereale) chromosomes using the restriction enzymes AluI, DraI, HpaII, and MspI.

Mitotic metaphase chromosomes of the rye inbred line L 301, which belongs to the Sortiment of the University of Hohenheim, were treated in situ with the restriction enzymes AluI (recognition sequence: 5′-AC/GT-3′), DraI (recognition sequence: 5′-TTT/AAA-3′), and the isoschizomeres HpaII and MspI (recognition sequence: 5′-C/CGG-3′) and stained with Giemsa. The chromosomes indicated similar banding patterns in comparison with the conventional Giemsa-C-banding. However, we have found in rye chromosomes after restrictase treatment that the telomeric bands were reduced in extension. In a lower degree the centromeric bands of individual chromosomes could be absent in dependence of the used restriction enzymes. The number of the intercalary bands were also reduced. Nevertheless, the tested restriction enzymes produced characteristic banding patterns of the rye genome. This uncomplicated banding technique is suited for a very quick banding method of karyotype analysis especially to obtain a first survey of the band patterns on the rye chromosomes.

Loss of nuclear DNA in leaves of rye.

The nuclear DNA content of rye leaf cells was cytophotometrically determined. At the commencement of differentiation nuclei in rye leaves remain standing at G1 phase. With further differentiation a remarkable diminution of nuclear DNA content occurs in diploid cells. The largest number of cells showing a loss of nuclear DNA content were found in the top of the leaf. The age of the leaf and the extent of diminution in nuclear DNA content are correlated.

Cytophotometrical measurement of nuclear DNA content in some coniferous and deciduous trees.

The DNA content of nuclei from meristematic root tip cells of five coniferous and one deciduous tree species and, for comparison, ofVicia faba was cytophotometrically determined. The DNA values of diploid nuclei fromGinkgo biloba are approximately a quarter lower than those fromVicia faba. The nuclear DNA values of the other tree species are merely a third to a ninth part of those ofVicia faba. In three tree species, as well as diploid, we have found nuclei of different polyploid level.The reliability of different cytochemical methods, which are used for determination of the nuclear DNA content, is critically analyzed. The DNA values of the investigated tree species are discussed in connection with the evolution of the DNA content in higher plants.

[The development of pollen grains and formation of pollen tubes in higher plants : V. The division of the generative nucleus in the pollen tube of Tradescantia paludosa]. / Untersuchungen zur Pollenentwicklung und Pollenschlauchbildung bei höheren Pflanzen : V. Zeitpunkt der Teilung des generativen Kerns in die beiden Spermakerne bei Pollenschlauchkulturen von Tradescantia paludosa.

Fixed pollen tubes of Tradescantia paludosa were investigated cytologically to determine the precise moment of division of the generative nucleus and to measure the length of the pollen tubes at the same time.The exact moment of division of the generative nucleus could be shown to be closely correlated with the length of the pollen tube of Tradescantia. In the majority of the pollen tubes the vegetative nuclei are not yet in the degenerative phase at the time of division of the generative nucleus. In all random samples investigated pollen tubes without nuclei were found. Depending on the random sample the percentage of pollen tubes without nuclei was 11-22%.In the discussion it is pointed out that the data obtained from the cytological investigations show that the findings of the precise moment of division of the generative nucleus agree with earlier published results on the development of pollen grains and formation of pollen tubes. The development of the male gametophyte of the angiosperms is to be divided into a series of stages. The data obtained here from the cyto-morphological studies of pollen tubes of Tradescantia and from cyto- and biochemical investigations of others indicate that the different stages of the pollen grain and the pollen tube can be separated. In the second part of the discussion there is presented an evaluation of the hypothesis that during evolution of the angiosperms species have formed repeatedly, and in most cases probably independently from one another, in which the degenerative phase of the vegetative nucleus begins earlier and earlier during the development of the male gametophyte, or that in some instances DNA replication is either partially or entirely eliminated.

[The development of pollen grains and formation of pollen tubes in higher plants : VI. Pollen grains of Tradescantia with two pollen tubes]. / Untersuchungen zur Pollenentwicklung und Pollenschlauchbildung bei höheren Pflanzen : VI. Pollenkörner mit zwei Pollenschläuchen bei der Gattung Tradescantia.

Pollen tube cultures of Tradescantia paludosa, Tradescantia virginiana and the hybrid Hutchinsonii (a cross of Tradescantia virginiana and andersoniana) were placed in vitro under various physiological conditions and analyzed cyto-morphologically in regard to double-tube formation.Depending on the culture conditions and on the plant variety studied there was found for Tradescantia to be a double-tube formation of 13% to 35%. Triple-tube formations as well as branching of the pollen tubes occurred in very rare exceptions. After a 1-2 hour addition of 0,02% colchicin solution to the nutritional medium a drastic reduction of the percentage of double-tubes was determined in the case of Tradescantia virginiana. A double-tube formation of only 6% was observed.In the case of double-tube formations both nuclei always immigrate into the same pollen tube. At the time of immigration the pollen tube with the nuclei is usually the longer of the two.For short growth times of up to 4 hours the averages lengths of both pollen tubes of a double-tube formation is significantly different. The growth of the two pollen tubes does not proceed synchronously. The pollen tube without the migrated nuclei ceases growth early in contrast to the tube containing the nuclei.A comparison of the averages lengths of double-tubes (after the lengths of both pollen tubes were added) with the corresponding values of single-tube pollen grains demonstrates that for experiments 1-5 (cultures under standard conditions and short growth times of up to 4 hours) there is no significant difference between the values. However the values of the double-tube pollen grains are substantially greater than those of the single-tube group in the case of experiment 6-8 (cultures under conditions varying from those of the standard and in some of them growth times longer than 4 hours). The values are significantly different. In experiments 1-5 with two exceptions there is a positive correlation between the averages lengths of the pollen tubes with the immigrated nuclei and those without.The possible causes for the formation of pollen grains with two pollen tubes in vitro are discussed. In the first section the question is debated what the reasons for the asynchronous growth of the two pollen tubes could be. In the second section the hypothesis is presented, that the double-tube formation as found in Tradescantia can be seen from the evolutionary viewpoint as a primitive characteristic. On the other hand the behavior of both nuclei immigrating into only one of the pollen tubes is hypothesized to be an advanced characteristic.

[A change in dominance of a mutation in barley, combined with suspension of the pleiotropic action of the gene]. / Ein fall von Dominanzwechsel bei gerste - zugleich ein beispiel für Aufhebung pleiotroper Genwirkungen?

In order to demonstrate change of dominance of mutants in altered genetic environments, a mutant of spring barley type Haisa II was used as the experimental material. This mutant is inherited as a single gene dominant. It differs from the original type by length of the grain, the rachis segment and the straw. The complex of characteristics of this mutant is pleiotropically inherited.Referring to these three characteristics the following plant types were analyzed: plants of the mutant, of the type Haisa II (from which the mutant is derived), seven other spring barley types serving as crossing partners of the mutant, and finally F1-plants of the MS-, MH- and SH-crossings.A change of dominance could be demonstrated in the case of the F1-plants of certain MS-crosses. With the F1-plants of several of these MS-crosses it could be shown, that the change of dominance is combined with suspension of pleiotropic gene action.In the discussion it is pointed out how little is known at the present time about the mechanisms which underlie a change of dominance and of pleiotropism. For a deeper understanding of these phenomena experiments on a moleculargenetic level, similar to those performed in other experimental systems, are necessary.

[The development of pollen grains and formation of pollen tubes in higher plants : I. Quantitative measurements of the DNA-content of generative and vegetative nuclei in the pollen grain and pollen tube of Petunia hybrida mutants]. / Untersuchungen zur Pollenentwicklung und Pollenschlauchbildung bei höheren Pflanzen : I. Quantitative Bestimmungen des DNS-Gehalts generativer und vegetativer Kerne in Pollenkörnern und Pollenschläuchen von Petunia-hybrida-Mutanten.

The DNA-content of generative and vegetative nuclei in mature pollen grains of four Petunia hybrida mutants was determined by cytophotometry. In addition the DNA-content of generative and vegetative nuclei in the pollen tube of two of these four mutants (virescens-2 n and ustulata-2 n) was cytophotometrically measured.The DNA-values found in the generative nuclei indicate that the DNA-replication continues in the mature pollen grain and comes to an end only after the migration of the nuclei into the pollen tube. These data are in disagreement with the results of DNA-measurements described for a limited number of other species which all show completion of DNA-synthesis during the maturation stage of the pollen grains.The vegetative nuclei of the four Petunia mutants studied show significant differences in the onset of the degenerative phase. Extreme variation is manifested in the ustulata-2 n mutant in which the degeneration of nuclei may reach the final stage in the maturing pollen grain. However in this mutant vegetative nuclei with an unaltered DNA-content may also be demonstrated in the pollen tube. Some of the vegetative nuclei in the pollen tube of ustulata-2 n exhibit an increased amount of DNA which could be the result of differential DNA-replication in the vegetative nuclei. The decrease of the DNA-content in a certain fraction of the vegetative nuclei in the maturing pollen grain does not agree with observations made in other species by several authors who report DNA constancy until the pollen grain is fully mature.The data obtained from the analysis of the four Petunia hybrida mutants point to an important role of the vegetative nucleus in the development of the pollen tube. The Petunia hybrida mutants may be regarded as especially favourable material for investigations concerning the function of the vegetative cell in the development of the pollen grain and pollen tube.