Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan^R) and -glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan^R calli. A high frequency of plants regenerated from Kan^R calli expressed both Kan^R and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.     

Trisomics from triploid-diploid crosses in self-incompatible Lycopersicum peruvianum

Summary An attempt was carried out to produce trisomics of the wild tomato L. peruvianum, to define their essential features, and to detect relationships between trisomy and the expression of self-compatibility.Triploid-diploid crosses in L. peruvianum yielded nearly 40% aneuploids. Of these, 18% were single trisomics, and the rest had 2, 3 and 4 extra chromosomes. Almost all the trisomics occurred in crosses where the triploid was used as female parent. Vigour and fertility of trisomics were not much different from those of disomics, and morphologically they were very similar.The extra chromosome was identified in three self-compatible trisomic plants through somatic and pachytene chromosome morphology. One of these plants was trisomic for chromosome 1, while the other two were trisomic for chromosome 3. In these trisomics a positive correlation was found between chromosome length and trivalent formation, but no relationship between chromosome length and frequency of laggards was observed.A series of test-crosses revealed that the capacity of the trisomics to produce seed upon selfing always resulted from alterations of the incompatibility phenotype of the style and not from competitive interaction in the pollen. Progeny analyses showed that the self-compatibility features of the trisomics were not transmitted from one generation to the next. The implications of these findings are discussed.This work has been supported by a contract between the European Communities and the CNEN. This publication is contribution no. 1458 from the Biology Division of the European Communities and contribution no. 472 from the Divisione Applicazioni delle Radiazioni del CNEN.     

Cellular and subcellular localization, N-terminal acylation, and calcium binding of Caenorhabditis elegans protein phosphatase with EF-hands

The RdgC/PPEF family of serine/threonine protein phosphatases is distinguished by the presence of C-terminal EF-hands and neuron-specific expression, including frequent expression in primary sensory neurons. Here we report that the sole Caenorhabditis elegans PPEF (CePPEF) homolog is also highly expressed in primary sensory neurons and is not found outside the nervous system. Neurons expressing CePPEF include the ciliary chemosensory neurons AWB and AWC; and within these neurons, CePPEF is highly enriched in the sensory cilia. In transgenic C. elegans and in transfected 293 cells, CePPEF is membrane-associated, and the N terminus of CePPEF is necessary and sufficient for this membrane association. [(3)H]Myristate and [(3)H]palmitate labeling studies in 293 cells demonstrated that this association was mediated by myristoylation at Gly(2) and palmitoylation at Cys(3). Introducing the G2A or C3S mutation into CePPEF greatly reduced membrane association in 293 cells and in transgenic nematodes. A recombinant C-terminal fragment of CePPEF containing two putative EF-hands bound between one and two Ca(2+) ions/protein, and mutation of residues presumed to ligand calcium in the two putative EF-hands led to diminished calcium binding. These results establish the first direct evidence for fatty acylation and calcium binding of a PPEF family member and demonstrate a remarkable conservation of sensory neuron expression among the members of this distinctive family of protein phosphatases.     

Synthesis and DNA-binding affinity of A-C8/C-C2 alkoxyamido-linked pyrrolo[2,1-c][1,4]benzodiazepine dimers

The synthesis of new A-C8/C-C2 alkoxyamido-linked pyrrolo[2,1-c][1,4]-benzodiazepine dimers have been described in this report. These dimers exhibit significant DNA-binding ability with moderate anticancer activity.     

Synthesis of C-8 alkylamino substituted pyrrolo[2,1-c][1,4]benzodiazepines as potential anti-cancer agents

The design and facile synthesis of C-8 alkylamino substituted pyrrolo[2,1-c][1,4]benzodiazepines is described. These have been prepared by linking the amines at C-8 position with propane spacer to improve solubility in water, and their in vitro cytotoxicity studies have been carried out.     

Early tapetal degeneration and meiotic defects are involved in the male sterility of Solanum commersonii (+) S. tuberosum somatic hybrids

 Somatic hybridization between Solanum commersonii and S. tuberosum resulted in the production of male-sterile hybrid plants, except for one fully male-fertile hybrid. The male-sterile hybrids exhibited a“pollen-less” phenotype, with rare pollen grains which were abnormal in shape and exine sculpture. Microsporogenesis and tapetal development were investigated both in male-sterile and male-fertile somatic hybrids to assess the cytological events that were involved in male sterility. The pattern of male sterility was complex, arising through mechanisms expressed at both sporophytic and gametophytic levels. Various abnormalities occurred first in the tapetum, and later during meiosis-II and cytokinesis. These caused the degeneration of the sporads and of the microspores when they were released. In the male-fertile hybrid, normal development of the tapetum and pollen mother cells was restored. The hypothesis that tapetal breakdown, meiosis-II and cytokinesis defects are related to each other, and depend on nuclear-mitochondrial interactions, is discussed. Because of the formation of multivalent chromosome configurations, it is likely that gene exchange between S. commersonii and S. tuberosum can occur in somatic hybrids, offering potential perspectives for the introgression of useful traits from S. commersonii into S. tuberosum. Received: 10 December 1996/Accepted: 21 March 1997     

Patterns of DNA and chromosome variation during in vitro growth in various genotypes of potato

Patterns of variation in nuclear DNA content and chromosome number were analysed in a temporal sequence, during in vitro growth of calli and cell suspensions in two monohaploids, a dihaploid and a tetraploid of potato (Solanum tuberosum). The results showed that both polyploidization and aneuploidy occurred during the initial stages of callus induction in all the genotypes. With further growth of callus, the frequency and extent of polyploidy and aneuploidy increased. In addition, the patterns of DNA and chromosome variation in cell suspension cultures revealed continued mitotic activity and transmission of cells with higher ploidy levels and aneuploidy. The results suggest that endoreduplication as well as endomitosis are important mechanisms of polyploidization, and that chromosome lagging and non-disjunction contribute to the production of aneuploidy.The various genotypes cultured under the same in vitro growth conditions differed in genetic instability, as assessed from the rate and degree of polyploidization and aneuploidy. Monohaploids showed more rapid rate of polyploidization than the dihaploid and tetraploid potatoes. It was concluded that the differences in genetic stability were due to different ploidy levels and genetic make-up of the genotypes.     

Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell–cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences.