The mouse lethal nonagouti (a(x)) mutation deletes the S-adenosylhomocysteine hydrolase (Ahcy) gene.

The lethal nonagouti (a(x)) mutation is a hypomorphic allele of the agouti coat color locus which, when homozygous, also leads to embryonic death around the time of implantation. To understand the molecular basis of these phenotypes, we identified and cloned a deletion breakpoint junction present in the ax chromosome. Long range restriction mapping demonstrated a simple deletion of approximately 100 kb, which does not affect agouti coding sequences, but begins only 4 kb 3' of the last exon, and thus may affect coat color by removing an agouti 3' enhancer. The Ahcy gene, which codes for the enzyme S-adenosylhomocysteine hydrolase (SAHase), is contained within a 20 kb region within the a(x) deletion. SAHase RNA and protein were detectable in early blastocysts and in embryonic stem cells, respectively, and analysis of embryos derived from an a(x)/a x a(x)/a embryo intercross indicated that a(x)/a embryos die between the late blastocyst and early implantation stages. Treatment of cultured embryos with an SAHase inhibitor, 3-deazaaristeromycin, or with metabolites that can result in elevated levels of cellular SAH, resulted in an inhibition of inner cell mass development, suggesting that loss of SAHase activity in a(x)/a(x) embryos is sufficient to explain their death around the time of implantation.     

Analysis of centromeric activity in Robertsonian translocations: implications for a functional acrocentric hierarchy

Approximately 90% of human Robertsonian translocations occur between nonhomologous acrocentric chromosomes, producing dicentric elements which are stable in meiosis and mitosis, implying that one centromere is functionally inactivated or suppressed. To determine if this suppression is random, centromeric activity in 48 human dicentric Robertsonian translocations was assigned by assessment of the primary constrictions using dual color fluorescence in situ hybridzation (FISH). Preferential activity/constriction of one centromere was observed in all except three different rearrangements. The activity is meiotically stable since intrafamilial consistency of a preferentially active centromere existed in members of six families. These results support evidence for nonrandom centromeric activity in humans and, more importantly, suggest a functional hierarchy in Robertsonian translocations with the chromosome 14 centromere most often active and the chromosome 15 centromere least often active.     

Competition between electron acceptors in photosynthesis: Regulation of the malate valve during CO2 fixation and nitrite reduction

For maximal rates of CO₂ assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO₂ fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO₂ fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO₂ fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the malate valve prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The malate valve is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the malate valve functions at various redox states of the acceptor side of Photosystem I.Abbreviations ARC anabolic reduction charge (NADPH/(NADP+NADPH)) - Chl chlorophyll - DTT dithiothreitol; Fd-ferredoxin - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetate - PS photosystem - qN non-photochemical quenching - qP photochemical quenching - _E quantum efficiency of PS II Dedicated to Prof. Dr. Hans Walter Heldt on the occasion of his 60th birthday.     

Different abilities of Friend murine leukemia virus (MuLV) and Moloney MuLV to induce promonocytic leukemia are due to determinants in both psi-gag-PR and env regions.

Moloney murine leukemia virus (M-MuLV) is capable of inducing promonocytic leukemia in 50% of adult BALB/c mice that have received peritoneal injections of pristane, but Friend MuLV strain 57 (F-MuLV) is nonleukemogenic under similar conditions. It was shown earlier that these differences could not be mapped to the U3 region of the virus long terminal repeat, indicating the probable influence of structural genes and/or R-U5 sequences. In this study, reciprocal chimeras containing exchanged structural genes and R-U5 sequences from these two closely related viruses were analyzed for differences in ability to induce disease. Results showed that two regions of F-MuLV, psi-gag-PR and env, when substituted for those of M-MuLV were dramatically disease attenuating. The 5'-most region, which is widely distributed, overlaps with the 5' end of the env intron and includes the RNA packaging region, psi, the entire gag coding region, and the viral protease coding region (PR) of pol. It was also found that reciprocal constructs having substitutions of both of these regions of M-MuLV in an F-MuLV background allowed full reestablishment of promonocytic leukemia. These leukemias were positive for c-myb rearrangements which are characteristic of M-MuLV-induced promonocytic leukemias. Neither region alone, however, was sufficient to produce disease with a greater incidence than 13%. Further studies demonstrated that the inability of viruses with psi, gag, PR, or env sequences from F-MuLV to induce leukemia in this model system was not due to their inability to replicate in hematopoietic tissue, to integrate into the c-myb locus early on after infection in vivo, or to express gag-myb mRNA characteristic of M-MuLV-induced preleukemic cells and acute leukemia.     

Interaction between fatty-acid and starch synthesis in isolated amyloplasts from cauliflower floral buds

The interaction of fatty-acid synthesis with starch synthesis has been studied in intact amyloplasts isolated from floral buds of cauliflower (Brassica oleracea L.). These amyloplasts perform acetate-dependent fatty acid synthesis at maximum rates only at high external ATP concentrations. Neither pyruvate nor malate inhibit acetate-dependent fatty-acid synthesis. In contrast, acetate is inhibitory to the low pyruvate-dependent fatty acid synthesis. These observations indicate that neither pyruvate nor malate are used as natural precursors of fatty-acid synthesis. In contrast to fatty-acid synthesis, the rate of glucose-6-phosphate-dependent starch synthesis is already saturated in the presence of much lower ATP concentrations. Rising rates of starch synthesis influence negatively the process of acetate-dependent fatty acid synthesis. This inhibition appears to occur under both limiting and saturating concentrations of external ATP, indicating that the rate of ATP uptake is limiting when both biochemical pathways are active. The rate of starch synthesis is modulated specifically by the concentration of 3-phosphoglycerate in the incubation medium. This observation leads to the conclusion that the activity of ADP-glucose pyrophosphorylase is of primary importance for the control of both, starch and fatty-acid synthesis. Using the modified approach of Kacser and Burns (1973; Symp. Soc. Exp. Biol.27, 65–104) we have quantified the contribution of the rate of starch synthesis to the control of the metabolic flux through fatty-acid synthesis.Abbreviations ADPGlc-PPase ADPglucose pyrophosphorylase - Glc6P glucose-6-phosphate - PGA 3-phosphoglyceric acid     

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement

Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM Arabidopsis medium - ANOVA analysis of variance - DAPI 4,6-Diamidino-2-phenylindole - PEG polyethyleneglycol     

Optimizing the generation of random amplified polymorphic DNAs in chrysanthemum

Many conditions of the RAPD reaction procedure may influence the result. This paper presents rapid detection of influential factors with a fractional factorial experiment. A more extensive study of these factors is also presented. Polymerase brand, thermal cycler brand, annealing temperature, and primer, are important factors in obtaining good DNA yields and optimal fragment patterns. Each primer has its optimal annealing temperature, and this is not correlated with the GC content of the primer. Optimal species-primer combinations have to be found by trial and error.