Enzymes of Choline Synthesis in Spinach (Response of Phospho-Base N-Methyltransferase Activities to Light and Salinity)

In spinach (Spinacia oleracea L.), choline is synthesized by the sequential N-methylation of phosphoethanolamine -> phosphomono- -> phosphodi- -> phosphotrimethylethanolamine (i.e. phosphocholine) followed by hydrolysis to release choline. Differential centrifugation of spinach leaf extracts shows that enzymes catalyzing the three N-methylations are cytosolic. These enzymes were assayed in leaf extracts prepared from plants growing under various light/dark periods. Under a diurnal, 8-h light/16-h dark photoperiod, the activity of the enzyme catalyzing the N-methylation of phosphoethanolamine is highest at the end of the light period and lowest following the dark period. Prolonged dark periods (exceeding 16 h) lead to a further reduction in the activity of this enzyme, although activity is restored when plants are reexposed to light. In contrast, the activity of the enzyme(s) catalyzing the N-methylations of phosphomono- and phosphodimethylethanolamine does not undergo comparable changes in response to light/dark treatments. Salt shock of plants with 200 mM NaCl results in a 2-fold increase in all three N-methylation activities relative to nonsalinized controls but only in plants exposed to light. Thus, light is required for the salt-responsive up-regulation of choline synthesis in spinach.     

The zygote and proembryo of alfalfa: quantitative,three-dimensional analysis and implications for biparental plastid inheritance

Summary Genetic studies have demonstrated biparental inheritance of plastids in alfalfa. The ratio of paternal to maternal plastids in the progeny varies according to the genotypes of the parents, which can be classified as strong or weak transmitters of plastids. Previous cytological investigations of generative cells and male gametes have provided no consistent explanation for plastid inheritance patterns among genotypes. However, plastids in the mature egg cells of a strong female genotype (6–4) were found to be more numerous and larger than in mature eggs of a weak female genotype (CUF-B), and the plastids in 6–4 eggs are positioned equally around the nucleus. In CUF-B, the majority of plastids are positioned below (toward the micropyle) the mid level of the nucleus, which is the future division plane of the zygote. Since only the apical portion of the zygote produces the embryo proper, plastids in the basal portion were predicted to become included in the suspensor cells and not be inherited. In the present study, we examined zygotes and a two-celled proembryo from a cross between CUF-B and a strong male genotype (301), a cross that results in over 90% of the progeny possessing paternal plastids only. Our results indicate that the distribution of plastids observed in the CUF-B egg cell is maintained through the first division of the zygote. Further, paternal plastids are similarly distributed; however, within the apical portion of the zygote and in the apical cell of the two-celled proembryo, the number of paternal plastids is typically much greater than the number of maternal plastids. These findings suggest that maternal and paternal plastid distribution within the zygote is a significant factor determining the inheritance of maternal and paternal plastids in alfalfa.     

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.     

Physical mapping of the human T-cell receptor beta gene complex,using yeast artificial chromosomes

Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human T-cell chain gene complex (TCRB). Variable region genes (BV) for the 25 known subfamilies were used as probes to screen the ICRF AM4x YAC library. Of the five positive YACs identified, one YAC designated B3, 820 kilobase pairs (kbp) in size, scored positive for all 25 TCRBV subfamilies plus the constant region genes (BC) when analyzed by pulse field gel electrophoresis. Restriction enzyme mapping of B3 located TCRBV and TCRBC gene regions to 4 Sfi I fragments of 280 110, 90, and 125 kbp and was in accordance with published data. In addition comparison of hybridization results of Sfi I-restricted B3 and genomic DNA from the parental cell line GM1416B revealed identical banding patterns. The data thus showed YAC B3 encoded a complete and unrearranged TCRB gene locus of some 600–620 kbp. The map was further resolved by locating restriction sites for Sal I and Bss HII on B3, giving more precise localization of the individual TCRBV gene families. Flourescent in situ hybridization of B3 to spreads of human metaphase chromosomes localized B3 to 7q35. However, two additional signals were obtained; one attributable to the TCRBV orphon cluster on 9p21, the second to the long arm of chromosome 2. Polymerase chain reaction amplification of a chromosome 2 somatic cell hybrid, using primers for all 25 TCRBV gene families, revealed that the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCRB locus flanked by chromosome 2 sequences.     

Soil microbial diversity and the sustainability of agricultural soils

Many world ecosystems are in various states of decline evidenced by erosion, low productivity, and poor water quality caused by forest clearing, intensive agricultural production, and continued use of land resources for purposes that are not sustainable. The biological diversity of these systems is being altered. Little research has been conducted to quantify the beneficial relationships between microbial diversity, soil and plant quality, and ecosystem sustainability. Ecosystem functioning is governed largely by soil microbial dynamics. Differences in microbial properties and activities of soils have been reported but are restricted to general ecological enumeration methods or activity levels, which are limited in their ability to describe a particular ecosystem. Microbial populations and their responses to stresses have been traditionally studied at the process level, in terms of total numbers of microorganisms, biomass, respiration rates, and enzyme activities, with little attention being paid to responses at the community or the organismal levels. These process level measurements, although critical to understanding the ecosystem, may be insensitive to community level changes due to the redundancy of these functions. As microbial communities comprise complex interactions between diverse organisms, they should be studied as such, and not as a black box into which inputs are entered and outputs are received at measured rates. Microbial communities and their processes need to be examined in relation to not only the individuals that comprise the community, but the effect of perturbations or environmental stresses on those communities.     

Resource polymorphisms in vertebrates

Discrete resource polymorphisms occur in various vertebrate species and probably occur more frequently than is generally appreciated. They are manifested in a number of ways, including morphological, behavioral and life history characters. Research on a number of unrelated taxa suggests that resource polymorphisms may be underestimated as a diversifying force and potentially play important roles in population divergence and initial steps in speciation. In an ecological context, they are important in resource partitioning and reducing intraspecific competition. Recent research suggests that the mechanisms maintaining these polymorphisms may be similar in diverse taxa, that phenotypic plasticity is important, and that some are under simple genetic control.     

Interactions between the terminal bases of mammalian introns are retained in inosine-containing pre-mRNAs.

Nuclear pre-mRNA splicing has a fundamentally similar two-step mechanism to that employed by group II self-splicing introns. It is believed that nuclear pre-mRNA splicing involves a network of RNA-RNA interactions which form the catalytic core of the active spliceosome. We show here a non-Watson-Crick interaction between the first and last guanosine residues of a mammalian intron. As in Saccharomyces cerevisiae, substitution of the conserved guanosines at the 5' and 3' splice sites by A and C respectively, specifically suppresses step 2 splicing defects resulting from the individual mutations. No other combination of terminal nucleotides was able to restore splicing. We additionally provide independent evidence for an indirect interaction between other nucleotides of the consensus splice sites during step 2 of splicing. Substitution of the nucleotide in the +3 position of the 5' splice site affects competition between closely spaced AG dinucleotides at the 3' splice site, although the interaction is not via direct differential base pairing. Finally, we show that complete substitution of guanosine residues by inosine in a pre-mRNA has only a modest effect upon step 2 of splicing, although earlier spliceosome assembly steps are impaired. Predictions can thus be made about the precise configuration of the non-Watson-Crick interaction between the terminal residues.