Production of somatic hybrids of moss by electrofusion

Summary Mixtures of protoplasts of two auxotrophic mutants of Physcomitrella patens, one requiring thiamine (aneurine), the other p-aminobenzoic acid, have been subjected to electrofusion. The protoplasts were aligned in an alternating electric field (500 KHz, 20 V RMS/cm) and induced to fuse by a brief DC pulse (800 V/cm, time constant lms). After culture, first on complete medium and then on selective medium, hybrid plants were obtained at a frequency of 3%. One hybrid with a morphology typical of polyploidy was also observed.Dedicated to Professor Georg Melchers to celebrate his 50-year association with the journal     

Hirsutinolides,glaucolides and sesquiterpene lactone from Vernonia species

The investigation of nine Vernonia species afforded in addition to known sesquiterpenes 28 new ones. The structures were elucidated by high field ¹H NMR spectroscopy and the configurations were determined by NOE difference spectroscopy and, in one case, by X-ray analysis. The results indicated that configurations of several previously reported sesquiterpene lactones have had to be revised. In addition to known types two new ones, the jalcaguaianolides and the vernojalcanolides, are described. Furthermore some unusual reaction products are presented which, in part, led to some natural occurring lactones.     

Isolation and cryopreservation of O-methylthreonine-resistant Rosa cell lines altered in the feedback sensitivity of l-threonine deaminase

O-Methylthreonine (OMT) inhibits the growth of plated Rosa cells (ID₅₀6·10^-6M). Isoleucine is able to reverse efficiently and specifically this OMT toxicity. From OMT-resistant colonies occurring at a frequency of 1.58·10^-7 variants per cell plated at 10^-4M OMT, the variant strains OMT^R-1 and OMT^R-2 were isolated, cloned via protoplasts and characterized. Both variants were ten times more resistant to OMT than the wildtype and were cross-resistant to another isoleucine analog, dl-4-thiaisoleucine. The resistant variants retained their resistance after storage for three years in liquid nitrogen. Both resistant strains were stable for several months when subcultured in the absence of OMT although it was shown in a reconstitution experiment that wildtype cells overgrow OMT^R-2 variant cells if co-cultivated for many passages in drug-free medium. One case of instability was observed upon long-term subculturing in drug-free medium: the strain OMT^R-1D^* partially lost phenotypic properties. Resistance to OMT was followed qualitatively by a new method based on inhibition-zone formation in cell suspensions plated in agar medium. The OMT-resistant variants showed a reduction in sensitivity of the enzyme l-threonine deaminase to feedback inhibition by isoleucine, a decreased stability of l-threonine deaminase when stored at-18°C or incubated at +55°C and a two- to threefold increase of the free isoleucine pool within the cells. The genetical events and the biochemical mechanisms which might lead to the observed stable and biochemically defined character are discussed with particular reference to the high ploidy level of the Rosa cell line.Abbreviations OMT l-O-methylthreonine - TD l-threonine deaminase     

The inter-relationship between heterochromatin distribution and chiasma distribution

The distribution of chiasmata and their relationship to the presence of fixed and polymorphic heterochromatic segments is described in seven grasshopper species. In six of these, Cryptobothrus chrysophorus, Trimerotropis bilobata, Calliptamus wattenwylianus, Arcyptera fusca, Pezotettix giorni and Acrotylus insubricus, the presence of terminally located polymorphic heterochromatic segments leads to a radical redistribution of chiasmata away from the segments to more proximal sites. Polymorphisms for proximal heterochromatic segments exist in the first three of these species and they lead to a predominance of terminally associated homologues at male meiosis. In Oxya japonica where both polymorphic and fixed blocks are present, the polymorphic blocks have a similar pronounced effect on chiasma distribution, whereas the fixed blocks have no such effect.     

Differences in regulatory sequences of naturally occurring JC virus variants.

The regulatory region was sequenced for DNAs representative of seven independent isolates of JC virus, the probable agent of progressive multifocal leukoencephalopathy. The isolates included an oncogenic variant (MAD-4), an antigenic variant (MAD-11), and two different isolates derived from the urine (MAD-7) and from the brain (MAD-8) of the same patient. The representative DNAs were molecularly cloned directly from diseased brain tissue and from human fetal glial cells infected with the corresponding isolated viruses. The regulatory sequences of these DNAs were compared with those of the prototype isolate, MAD-1, sequenced previously (R. J. Frisque, J. Virol. 46:170-176, 1983). We found that the regulatory region of JC viral DNA is highly variable due to complex alterations of the previously described 98-base-pair repeat of MAD-1 DNA. On the basis of these alterations, there are two general types of JC virus. There were no consistent alterations in regulatory sequences which could distinguish brain tissue DNAs from tissue culture DNAs. Furthermore, for each isolate except MAD-1 (R. J. Frisque, J. Virol. 46:170-176, 1983), the regulatory regions of brain tissue and tissue culture DNAs were not identical. The arrangement, sequence, or both of potential regulatory elements (TATA sequence, GGGXGGPuPu, tandem repeats) of JC viral DNAs are sufficiently different from those in other viral and eucaryotic systems that they may effect the unique properties of this slow virus.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

In vitro development of the hamster and chick secondary palate

A series of experiments were undertaken to compare the in vitro behaviour of the medial edge epithelium (MEE) of hamster, in which palatal shelves normally fuse, and chick, in which they do not fuse. Homotypic pairs of hamster and chick embryo palatal processes, single palatal processes, and heterotypic palatal shelves of both animals were grown in vitro. The results indicated that contact between palatal shelves may not be crucial for MEE differentiation in mammals. The ability to acquire pre-fusion characteristics may be present in mammalian palatal tissue from their early development and may be expressed by cessation of DNA synthesis in the MEE, elevation of cAMP, and MEE cell death. Isolated chick palatal shelf cultured under identical conditions did not express these mammalian pre-fusion characteristics. When MEE of hamster and chick palatal shelves were placed in contact with one another, the intervening epithelia underwent cytolysis. This could be due to either the destruction of chick MEE by lysosomal enzymes liberated from adjacent degenerating hamster MEE cells, or by induction of cell death in chick MEE by hamster mesenchyme. Heterotypic palatal tissue combinations also suggest that release of lysosomal enzymes in the hamster MEE, which leads to its dissolution, may be the terminal event in epithelial differentiation prior to the establishment of mesenchymal continuity. It is suggested that an inverse relationship exists between DNA synthesis and cAMP levels during palatogenesis: when palate closes (as in mammals) the MEE is eliminated by increasing cAMP levels, whereas when palate remains open (as in birds) low level of cAMP preserve the integrity of MEE by supporting DNA synthesis.     

Concordant variation in thermal tolerance and allozymes of the red shiner,Notropis lutrensis,inhabiting tailwater sections of the Brazos River,Texas

Synopsis Critical thermal maxima (CTM) and genetic variation were compared for red shiners, Notropis lutrensis, from regulated and unregulated sites on the Brazos River in northcentral Texas. Tailwater fish acclimated to 25°C had significantly lower CTM's than those from a site upstream from the dam and unregulated downstream sites. Significantly different intrasite variances were observed, with two- and four-fold larger CTM variances in fish from within 1 km and 30 km of the dam. Genetic variation was determined from electrophoretic comparisons at 21 structural gene loci. Mean heterozygosity was greatest at regulated sites. Tests for locus heterogeneity at five variable loci indicated that regulated and unregulated populations are not homogeneous. Fish under regulation were genetically more similar to each other than they were to those not affected by regulation. The proportions of the gene variance attributable to habitat alteration were partitioned, and fully one-third of the gene variation was attributed to stream regulation. Patterns of variation in thermal tolerance and metabolic enzymes in the red shiner correlated closely with temperature regimes associated with hypolimnion release from the dam. These adaptive responses have occurred in less than 40 years.