Thermostabilization of protein A-luciferase fusion protein by single amino acid mutation

A gene expression plasmid, pMALU7, for coding a fusion protein between protein A (SpA) and mutated firefly luciferase (Luc), was constructed. The fused gene was expressed in Escherichia coli and the resulting protein (SpA-LucTS) was purified with affinity chromatography. By changing a single amino acid, from Glu to Lys at the position 354 in the luciferase moiety, the thermostability of luciferase was improved.     

Predictability of the terrestrial carbon cycle

Terrestrial ecosystems sequester roughly 30% of anthropogenic carbon emission. However this estimate has not been directly deduced from studies of terrestrial ecosystems themselves, but inferred from atmospheric and oceanic data. This raises a question: to what extent is the terrestrial carbon cycle intrinsically predictable? In this paper, we investigated fundamental properties of the terrestrial carbon cycle, examined its intrinsic predictability, and proposed a suite of future research directions to improve empirical understanding and model predictive ability. Specifically, we isolated endogenous internal processes of the terrestrial carbon cycle from exogenous forcing variables. The internal processes share five fundamental properties (i.e., compartmentalization, carbon input through photosynthesis, partitioning among pools, donor pool‐dominant transfers, and the first‐order decay) among all types of ecosystems on the Earth. The five properties together result in an emergent constraint on predictability of various carbon cycle components in response to five classes of exogenous forcing. Future observational and experimental research should be focused on those less predictive components while modeling research needs to improve model predictive ability for those highly predictive components. We argue that an understanding of predictability should provide guidance on future observational, experimental and modeling research.     

拟南芥PKA催化亚基基因的克隆、原核表达及其催化活性的鉴定

蛋白质的磷酸化与脱磷酸化是生物体内存在的一种普遍的调节方式，几乎参与所有的生命活动过程。利用Blast2.0分析拟南芥基因组序列发现存在一个与动物蛋白激酶cDNA同源性的序列。在GenBank中比较发现它与动物的依赖cAMP的蛋白激酶（PKA）的催化亚基（C亚基）有相似的特征序列。提取拟南芥（Arabidopsis thaliana (L.)Heynh.)的总ＲＮＡ，通过ＲＴ－ＰＣＲ克隆得到这一cDNA片段，经序列测定证实它具有完整的阅读框架，将其克隆至pET30a原核表达载体，结果表明在大肠杆菌（E.coli)BL21(DE3)中该表达质粒在IPTG诱导下表达产生大量带寡聚组氨酸标记的重组蛋白，该蛋白在37℃表达时主要以包含体形式存在，而在22℃表达时主要以可溶性蛋白形式存在，经过与组氨酸结合金属螯合树脂亲和柱层析纯化后，得到纯化的目的蛋白，其纯度达到87%以上。活性鉴定表明其具有依赖于cAMP的蛋白激酶活性。而加入PKA的抑制剂（H-8）后，其活性显著下降。从而证实它确实是拟南芥的PKA催化亚基。Western blot结果显示它几乎不受ABA，NaCl等逆境的诱导。     

The olfactory G protein Gαolf possesses a lower GDP-affinity and deactivates more rapidly than Gsαshort: consequences for receptor-coupling and adenylyl cyclase activation

The olfactory G protein G(alphaolf) differs from the short splice variant of G(salpha) (G(salphaS)) in 80 amino acids, but little is known about biochemical differences between G(alphaolf) and G(salphaS). We addressed this question by analyzing fusion proteins of the beta2-adrenoceptor (beta2AR) and G(alphaolf) and G(salphaS), respectively, using Sf9 insect cells as expression system. The fusion ensured defined receptor/G protein stoichiometry and efficient coupling. High-affinity agonist binding studies showed that G(alphaolf) possesses a lower GDP-affinity than G(salphaS) As a result, the agonist-free beta2AR and the beta2AR occupied by partial agonists were more efficient at promoting GDP-dissociation from G(alphaolf) than from G(salphaS) a assessed by guanosine 5'-O-(3-thiotriphosphate) binding, adenylyl cyclase (AC) activity and GTP hydrolysis. Basal AC activity in the absence of GTP was almost sixfold lower in membranes expressing beta2AR-G(alphaolf) than in membranes expressing beta2AR-G(salphaS) at similar levels, reflecting the lower abundance of G(alphaolf-GDP) relative to G(salphaS-GDP). The maximum agonist-stimulated AC activity with beta2AR-G(salphaS) was more than twofold higher than with beta2AR-G(alphaolf), but the relative agonist-stimulation of AC with beta2AR-G(alphaolf) was much greater than with beta2AR-G(salphaS). The difference in maximum AC activity can be explained by more rapid deactivation of G(alphaolf-GTP) by GTP hydrolysis and GTP dissociation relative to G(salphaS-GTP). Taken together, there are biochemical differences between G(alphaolf) and G(salphaS), supporting different roles of these G proteins in vivo.     

Nuclear and cell fusion cause polyploidy in the megagametophyte of common cypress, Cupressus sempervirens L.

In common cypress, Cupressus sempervirens L., the megagametophyte persists in mature seeds as a polyploid endosperm containing cells with even and odd series of DNA contents: 1C, 2C, 3C, 4C, 5C etc., where C is the amount of DNA in the haploid genome. In this study, cytometrical, histological and cytochemical investigations were performed in order to determine the behavior of megagametophyte nuclei during the reproductive cycle. Unexpected nuclear alterations due to a continuous process of nuclear fusion were observed in the megagametophyte, leading to polyploidization and consequently to intense food-reserve synthesis. During the free nuclear stage, the megagametophyte exhibited only sporadic nuclear fusion and limited food-reserve production. When cellularization took place, multinucleated compartments were observed in which nuclei fused, producing odd and even series of DNA contents as proved by flow-cytometric analysis. This polyploidization process considerably increased after fertilization and during embryo development, and was accompanied by increased food-reserve synthesis. During these later stages, fusion mainly involved nuclei of contiguous cells and was preceded by the disintegration of their adjacent walls. Mitoses with incomplete phragmoplast differentiation were also observed to yield polyploid nuclei. Finally, in mature seeds the endosperm still exhibited multinucleate cells and fusion nuclei, and contained high amounts of storage products. The results are interpreted as an alteration of DNA contents in the megagametophyte cells in relation to specific metabolic activity during seed development. Received: 2 September 1998 / Accepted: 31 December 1998     

Subject index volume 38 (1985)

Reproducibly high yields of protoplasts were obtained from the unicellular asexual green alga Chlorella saccharophila (Krüger) Nadson 211-1a by treating the cells with polysaccharide degrading enzymes. A maximum of 32% of the protoplasts were able to regenerate cell walls and formed colonies when plated on a regeneration medium. Chlorophyll deficient mutants were isolated after X-ray irradiation of the green wild-type cells. These mutants differed in growth requirements and light sensitivity from each other and from the wild-type cells. Intraspecific protoplast fusion of a yellow with a white pigment mutant strain was accomplished by polyethylene glycol (PEG) and Ca²⁺ and green hybrid cells were obtained using selective conditions. Ultrastructural and morphological investigations carried out so far demonstrate differences between hybrid cells and the parental mutant cells as well as between hybrid cells and the green wild-type cells.     

Asymmetric somatic hybrids between Lycopersicon esculentum and irradiated Lycopersicon peruvianum

Summary Asymmetric somatic hybrids of Lycopersicon esculentum and Lycopersicon peruvianum were obtained by fusion of leaf protoplasts from both species after irradiation of protoplasts or leaf tissue of L. peruvianum with 50, 300, or 1,000 Gy of gamma-rays. These radiation doses were sufficient to abolish the growth of the L. peruvianum protoplasts. The hybrids were selected for their ability to regenerate plants; this regeneration capacity derived from L. peruvianum. All asymmetric hybrid plants were aneuploid. The ploidy level, the morphology, and the regeneration rate were analyzed in relation to the radiation dose applied to L. peruvianum. After a low dose (50 Gy), most hybrids had near-triploid chromosome numbers, whereas after a high dose (300 or 1,000 Gy), most hybrids had near-pentaploid numbers. The morphology of the asymmetric hybrids was intermediate between that of L. esculentum and symmetric somatic hybrids of both species (obtained without irradiation treatment), and approached the morphology of L. esculentum to a greater extent after a high dose of irradiation. The asymmetric hybrids regenerated more slowly than the symmetric hybrids and regeneration proceeded more slowly after a high dose than after a low dose of irradiation. The high-dose hybrids also grew more slowly, flowered less, and set fruits less than the low-dose hybrids. No seeds could be obtained from any asymmetric hybrid.     

Fertile somatic hybrids of Solanum species: RFLP analysis of a hybrid and its sexual progeny from crosses with potato

Summary Restriction fragment length polymorphism (RFLP) markers were used to distinguish the chromosomes of Solanum brevidens from those of potato (S. tuberosum) in a fertile somatic hybrid. The hybrid had markers that account for all 24 chromosome arms from each parent, indicating that the hybrid contained at least one copy of each chromosome from each parent. The markers were then used to follow segregation of chromosomes in sexual progeny that resulted from a cross of the somatic hybrid with the potato cultivar Katahdin. Approximately 10% of the sexual progeny lacked one or more of the markers specific to S. brevidens. No one chromosome or marker appeared to be lost preferentially. This infrequent absence of a chromosome marker derived from the wild parent could be explained by intergenomic pairing and recombination. The loss of a marker band for chromosome 8, coupled with the retention of two flanking markers, suggested that a small region of DNA was deleted during regeneration of the somatic hybrid. These results show the value of RFLP analysis when applied to somatic hybrids and their progeny. Clearly, RFLPs will be useful for following the DNA from wild species during its introgression into potato cultivars.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Selection and characterization of somatic hybrids between Lycopersicon esculentum and Lycopersicon peruvianum

Somatic hybrids of the cultivated tomato, Lycopersicon esculentum, and a wild species, L. peruvianum, were obtained by fusion of leaf protoplasts from both species in the presence of poly-ethylene-glycol (PEG) or in an electric field. The somatic hybrids were selected on the basis of kanamycin resistance of L. esculentum and the plant regeneration capacity of L. peruvainum. Chromosome counts in root tips and the determination of the number of chloroplasts in guard cell pairs revealed that the majority of these hybrids was tetraploid (2n = 4x = 48). The remaining hybrids were at the hexaploid level with chromosome numbers between 64 and 72. The hybrid nature of the regenerated plants was confirmed by analysis of isozyme markers and by their morphology. Most hybrids did flower and set fruits and seeds after selfing. According to RFLP analysis 6 out of the 10 hexaploid hybrids contained two genomes of L. esculentum and four genomes of L. peruvianum. One of these hexaploids had genomes of two different L. peruvianum genotypes and was therefore considered to be derived from a triple protoplast fusion. The hexaploid plants were less fertile than the tetraploids and more resembled L. peruvianum.