The distribution of Escherichia coli recA protein bound to duplex DNA with single-stranded ends

A short single-stranded tail on one end of an otherwise duplex DNA molecule enables recA protein, in the presence of ATP and MgCl2, to form a complex with the DNA which extends into the duplex portion of the molecule. Nuclease protection studies at a concentration of MgCl2 which permits homologous pairing showed that cleavage by restriction endonucleases at sites throughout the duplex region was inhibited, whereas digestion by DNase I was not affected. These results indicate that recA protein binds to the duplex portion of tailed DNA allowing access by DNase I to a random sample of the many sites at which it cleaves, but providing limited protection of the relatively rare restriction sites. Electron microscopy revealed that the recA nucleoprotein complex with duplex DNA is indeed a segmented or interrupted filament that, with time, extends further from the single-stranded tail into the duplex region. recA protein binding extended into the duplex region more rapidly for duplexes with 5' tails than for those with 3' tails. These observations show that recA protein translocates from a single-stranded region into duplex DNA in the form of a segmented filament by a mechanism that is not strongly polarized.     

Assay of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.     

Inhibition of Threonine Dehydratase Is Herbicidal

Threonine dehydratase, the first enzyme in isoleucine biosynthesis, catalyzes deamination and dehydration of threonine to produce 2-ketobutyrate and ammonia. An antimetabolite, 2-(1-cyclohexen-3(R)-yl)-S-glycine (CHG), inhibits the plant enzyme. CHG inhibits the growth of Black Mexican Sweet corn (Zea mays) cells and of Arabidopsis thaliana plants. The herbicidal effects of CHG can be reversed by 2-ketobutyrate, other intermediates of isoleucine biosynthesis, and by isoleucine itself. These results suggest that the herbicidal effects observed with CHG are a consequence of inhibition of threonine dehydratase. The enzyme could be a potential target site for an herbicide screening program.     

Identification and Characterization of a Biodegradative Form of Threonine Dehydratase in Senescing Tomato (Lycopersicon esculentum) Leaf

Threonine dehydratase (TD; EC.4.2.1.16) is a key enzyme involved in the biosynthesis of isoleucine. Inhibition of TD by isoleucine regulates the flow of carbon to isoleucine. We have identified two different forms of TD in tomato (Lycopersicon esculentum) leaves. One form, present predominantly in younger leaves, is inhibited by isoleucine. The other form of TD, present primarily in older leaves, is insensitive to inhibition by isoleucine. Expression of the latter enzyme increases as the leaf ages and the highest enzyme activity is present in the old, chlorotic leaves. The specific activity of the enzyme present in older leaves is much higher than the one present in younger leaves. Both forms can use threonine and serine as substrates. Whereas TD from the older leaves had the same Km (0.25 mM) for both substrates, the enzyme from the young leaves preferred threonine (Km = 0.25 mM) over serine (Km = 1.7 mM). The molecular masses of TD from the young and the old leaves were 370,000 and 200,000 D, respectively. High levels of the isoleucine-insensitive form of threonine dehydratase in the older leaves suggests an important role of threonine dehydratase in nitrogen remobilization in senescing leaves.     

Inhibition of ion accumulation in maize roots by abscisic acid

Summary An inhibition of root growth, a decrease in the amount of potassium (as ⁸⁶Rb) and phosphate (³²P) accumulation by the root, and a partial depolarization of transmembrane electropotential were observed to develop with a similar time course and to a similar extent when intact maize (Zea mays L.) roots were treated with 10^-5 M abscisic acid (ABA). Potassium uptake was inhibited by ABA when excised, low-salt roots were bathed in KCl, KH₂PO₄, or K₂SO₄. ABA did not affect the ATP content of the tissues, the activity of isolated mitochondria, nor the activity of microsomal K⁺-stimulated ATPases.     

Under how wide a set of conditions will nonrandom mating occur in Raphanus sativus (Brassicaceae)?

Studies of the weedy annual Raphanus sativus have demonstrated that nonrandom mating, a prerequisite for sexual selection, can occur in greenhouse plants. To determine whether this nonrandom mating pattern can occur under a wide range of conditions, including conditions that might occur in the field, we considered variation in both maternal condition and pollen load size. Maternal condition was varied by altering the watering regime. Pollen load size was varied from approximately 26 to 343 pollen grains per stigma. At the smallest pollen load size, patterns of seed paternity were altered in two of the three pollen donor pairs; seed paternity became more equal among donors. For one of three pollen donor pairs, seed paternity was more divergent among donors on stressed maternal plants. Finally, for one pollen donor pair, rank order of pollen donor performance changed from the medium to the small pollen loads on stressed vs. control maternal plants. Thus, some field conditions may alter patterns of nonrandom mating in wild radish.     

Pollen performance of Raphanus sativus (Brassicaceae) declines in response to elevated [CO2]

Although increases in atmospheric [CO₂] are known to affect plant physiology, growth and reproduction, understanding of these effects is limited because most studies of reproductive consequences focus solely on female function. Therefore, we examined the effects of CO₂ enrichment on male function in the annual Raphanus sativus. Pollen donors grown under elevated [CO₂] initially sired a higher proportion of seeds per fruit than ambient [CO₂]-grown plants when each was tested against two different standard competitors; however, by the end of the 5-month experiment, these pollen donors sired fewer seeds than ambient [CO₂]-grown plants and produced a lower proportion of viable pollen grains. The results of this experiment confirm that elevated [CO₂] can alter reproductive success. Additionally, the change in response to elevated [CO₂] over time varied among pollen donor families; thus, changes in [CO₂] could act as a selective force on this species.     

Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.     

Novel quantitative trait loci (QTL) for Fusarium head blight resistance in wheat cultivar Chokwang

Fusarium head blight (FHB) is one of the most destructive diseases in wheat. This study was to identify new quantitative trait loci (QTL) for FHB resistance and the molecular markers closely linked to the QTL in wheat cultivar Chokwang. The primers of 612 simple sequence repeats (SSRs) and 12 target-region-amplified polymorphism (TRAP) marker were analyzed between resistant (Chokwang) and susceptible (Clark) parents. One hundred and seventy-two polymorphic markers were used to screen a population of 79 recombinant inbred lines (RILs) derived from the cross of Chokwang and Clark. One major QTL, Qfhb.ksu-5DL1, was identified on chromosome 5DL. The SSR marker Xbarc 239 was mapped in the QTL region, and also physically located to the bin of 5DL1-0.60-0.74 by using Chinese Spring deletion lines. Another QTL Qfhb.ksu-4BL1was linked to SSR Xbarc 1096 and tentatively mapped on 4BL. A QTL on 3BS, Qfhb.ksu-3BS1, was also detected with marginal significance in this population. Different marker alleles for these QTL were detected between Chokwang and Sumai 3 and its derivatives. These results suggested that Chokwang contains new QTL for FHB resistance that are different from those in Sumai 3. Pyramiding resistance QTL from various sources may enhance FHB resistance in wheat cultivars.