Molecular Cloning and Sequence of Potato Granule-Bound Starch Synthase Gene

It is known that tuber-specific expressions of many genes exist in the process of tuber development from stolon in potato (Solanum tuberosum). Study on the regulation of those gene expression will share light on the mechanism of organ-specific gene expression. Potato GBSS (granule-bound starch synthase) gene, which is solely responsive for the pres- ence of amylose in potato tuber, expression is tuber-specific. The paper describes the construction of a genomic library of a Chinese potato cultivar "Dongnong 303" in which 20 clones were isolated using partial GBSS gene sequence ampified by PCR. 5428 bp DNA sequence of one clone (GBSS17-1) was determined, including 1823 bp 5' flanking region. 2964 bp structure gene, and 641 bp 3' flanking region. It is highly homologious with reported GBSS gene sequence. In addition, the 730 bp most upstream sequence of 5' flanking region which was not reported previously contained stem and loop structures. The present result may provide some important information for further study in the molecular mechanism of organ specific gene expression.     

Biochemical Evidence for the Role of the Waxy Protein from Pea (Pisum sativum L.) as a Granule-Bound Starch Synthase

Proteins were solubilized from starch extracted from developing pea (Pisum sativum L.) embryos and chromatography of these proteins on a Mono-Q column separated two peaks of starch synthase activity. The major activity peak comprised more than 80% of the total activity. This fraction contained only the Waxy protein, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by staining for proteins or by immunoblot. A 77-kD polypeptide associated with the starch granules and presumed by others to be a starch synthase could not be detected in any of the active fractions. The native molecular weight of the solubilized starch synthase was 59,600 [plus or minus] 1700 as determined by sucrose density gradient. It is concluded that in pea seeds the Waxy protein and the starch synthase bound to the granule are the same protein.     

Cloning and Characterization of the Wx Gene Encoding a Granule-Bound Starch Synthase in Lotus (Nelumbo nucifera Gaertn)

The granule-bound starch synthase (GBSS) proteins were widely considered as one of the most important enzymes in plant amylose synthesis. However, understanding of the molecular basis of the GBSS protein in lotus remains fragmented. In this work, a lotus Wx gene, encoding a GBSS (GenBank accession no. EU938541), was isolated and characterized. This gene comprises 13 exons and 12 introns and covers 4152?bp (GenBank accession no. FJ602702). The exons of Wx gene have similar lengths, while the introns vary greatly. Phylogenetic tree indicated that the lotus GBSS protein belongs to a GBSS I subgroup. The expression of the Wx gene varies in different organs of the lotus during its development process and is also expressed differently in different cultivars. The Wx gene is expressed at a higher level in the rhizomes of cultivar Meirenhong than in those of cultivar Elian 4. This study elucidates more molecular information about the Wx gene in lotus and provides a theoretical foundation for the genes regulation and the modification of starch quality.     

Formation and Deposition of Amylose in the Potato Tuber Starch Granule Are Affected by the Reduction of Granule-Bound Starch Synthase Gene Expression

The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via antisense RNA in Agrobacterium rhizogenes-transformed potato plants. Analysis of starch production and starch granule composition in transgenic tubers revealed that reduction of GBSS activity always resulted in a reduction of the production of amylose. Field experiments, performed over a 2-year period, showed that stable inhibition of GBSS gene expression can be obtained. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. In plants showing inhibition of GBSS gene expression, the reduced amylose content in tuber starch was not a consequence of a lower amylose content throughout the entire starch granule. Starch granules of transgenic tubers were found to contain amylose at a percentage similar to wild-type starch in a core of varying size at the hilum of each granule. This indicated that reduced GBSS gene expression results in amylose formation in a restricted zone of the granules. The size of this zone is suggested to be dependent on the GBSS protein level. During development of the granules, the available GBSS protein is thought to become limiting, resulting in the formation of starch that lacks amylose. RNA gel blot analysis of tuber tissue showed that inhibition of GBSS gene expression resulted in a reduced GBSS mRNA level but did not affect the expression level of other starch synthesizing enzymes. Antisense RNA could only be detected in leaf tissue of the transgenic plants.     

The Promoter of the Gene for Plastidic Glutamine Synthetase (GS2) fromRice Is Developmentally Regulated and Exhibits Substrate-Induced Expression in Transgenic Tobacco Plants

A 1.3-kb fragment from the 5'-flanking region of the RGS-38gene, which encodes the plastidic glutamine synthetase in Oryzasativa L., was fused to a ß-glucuronidase (GUS) reportergene and introduced into Nicotiana tabacum by Agrobacterium-mediatedtransformation. The promoter directed GUS expression, both inleaves and in roots, and the expression of GUS was regulatedby light. The GUS activity was high in the mature leaves ofthe transgenic tobacco plants, in marked contrast to the activityof the GS1 promoter. The GS2 promoter also responded to externallyapplied ammonia, as is the case for the GS1 promoter. Theseresults suggest that the cis-acting regulatory elements thatcontrol the response to ammonia, a substrate for glutamine synthetase,are located within a 1.3-kb region of the promoter. (Received October 1, 1991; Accepted January 20, 1992)     

The Expression of Three Opsin Genes from the Compound Eye of Helicoverpa armigera (Lepidoptera: Noctuidae) Is Regulated by a Circadian Clock,Light Conditions and Nutritional Status

Visual genes may become inactive in species that inhabit poor light environments, and the function and regulation of opsin components in nocturnal moths are interesting topics. In this study, we cloned the ultraviolet (UV), blue (BL) and long-wavelength-sensitive (LW) opsin genes from the compound eye of the cotton bollworm and then measured their mRNA levels using quantitative real-time PCR. The mRNA levels fluctuated over a daily cycle, which might be an adaptation of a nocturnal lifestyle, and were dependent on a circadian clock. Cycling of opsin mRNA levels was disturbed by constant light or constant darkness, and the UV opsin gene was up-regulated after light exposure. Furthermore, the opsin genes tended to be down-regulated upon starvation. Thus, this study illustrates that opsin gene expression is determined by multiple endogenous and exogenous factors and is adapted to the need for nocturnal vision, suggesting that color vision may play an important role in the sensory ecology of nocturnal moths.     

Cellular Expression and Proteolytic Processing of Presenilin Proteins Is Developmentally Regulated During Neuronal Differentiation

Abstract: We have determined the expression of the Alzheimer's disease-associated proteins presenilin-1 and presenilin-2 in primary cultures of rat hippocampal neurons. Neurons highly express presenilin-1 and presenilin-2, whereas both proteins were not detected in astrocytes. Further, we have analyzed the subcellular localization and expression in rat hippocampal neurons during development. Although presenilin proteins were localized predominantly to the endoplasmic reticulum in nonneuronal cells transfected with presenilin cDNAs, in neurons, presenilin proteins were also found in compartments not staining with antibodies to grp78(BiP). Presenilin-1 and presenilin-2 were predominantly detected in vesicular structures within the somatodendritic compartment with much less expression in axons. Polarized distribution of presenilin-1 and presenilin-2 differs slightly, with more presenilin-2 expressed in axons compared with presenilin-1. Presenilin expression was found to be developmentally regulated. Presenilin expression strongly increased during neuronal differentiation until full morphological polarization and then declined. No full-length presenilin-1 or presenilin-2 could be detected within cell lysates. At early developmental stages the expected ～34-kDa N-terminal proteolytic fragment of presenilin-1 and the ～38-kDa fragment of presenilin-2 were detected. Later during differentiation we predominantly detected a ～38-kDa fragment for presenilin-1 and a ～42-kDa fragment for presenilin-2. By epitope mapping, we show that these slower migrating peptides represent N-terminal proteolytic fragments, cleaved C-terminal to the conventional site of processing. It is noteworthy that both presenilin-1 and presenilin-2 undergo alternative proteolytic cleavage at the same stage of neuronal differentiation. Regulation of presenilin expression and proteolytic processing might have implications for the pathological as well as the biological function of presenilins during aging in the human brain.     

Developmentally Regulated Expression of the Gene Family for Cytosolic Glutamine Synthetase in Pisum sativum

In Pisum sativum, two classes of genes encode distinct isoforms of cytosolic glutamine synthetase (GS). The first class comprises two nearly identical or “twin” GS genes (GS341 and GS132), while the second comprises a single GS gene (GS299) distinct in both coding and noncoding regions from the “twin” GS genes. Gene-specific analyses were used to monitor the individual contribution of each gene for cytosolic GS during root nodule development and in cotyledons during germination, two contexts where large amounts of ammonia must be assimilated by GS for nitrogen transport. mRNAs corresponding to all three genes for cytosolic GS were shown to accumulate coordinately during a time course of nodule development. All the GS mRNAs also accumulate to wild-type levels in mutant nodules formed by a nifD⁻ strain of Rhizobium leguminosarum indicating that induced GS expression in pea root nodules does not depend on the production of ammonia. Distinct patterns of expression for the two classes of GS genes were observed in certain mutant root nodules and most dramatically in cotyledons of germinating seedlings. The different patterns of expression between the two classes of genes for cytosolic GS suggests that their distinct gene products may serve nonoverlapping functions during pea development.     

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.     

Circadian Clock Gene Per2 Is Not Necessary for the Photoperiodic Response in Mice

In mammals, light information received by the eyes is transmitted to the pineal gland via the circadian pacemaker, i.e., the suprachiasmatic nucleus (SCN). Melatonin secreted by the pineal gland at night decodes night length and regulates seasonal physiology and behavior. Melatonin regulates the expression of the β-subunit of thyroid-stimulating hormone (TSH; Tshb) in the pars tuberalis (PT) of the pituitary gland. Long day-induced PT TSH acts on ependymal cells in the mediobasal hypothalamus to induce the expression of type 2 deiodinase (Dio2) and reduce type 3 deiodinase (Dio3) that are thyroid hormone-activating and hormone-inactivating enzymes, respectively. The long day-activated thyroid hormone T₃ regulates seasonal gonadotropin-releasing hormone secretion. It is well established that the circadian clock is involved in the regulation of photoperiodism. However, the involvement of the circadian clock gene in photoperiodism regulation remains unclear. Although mice are generally considered non-seasonal animals, it was recently demonstrated that mice are a good model for the study of photoperiodism. In the present study, therefore, we examined the effect of changing day length in Per2 deletion mutant mice that show shorter wheel-running rhythms under constant darkness followed by arhythmicity. Although the amplitude of clock gene (Per1, Cry1) expression was greatly attenuated in the SCN, the expression profile of arylalkylamine N-acetyltransferase, a rate-limiting melatonin synthesis enzyme, was unaffected in the pineal gland, and robust photoperiodic responses of the Tshb, Dio2, and Dio3 genes were observed. These results suggested that the Per2 clock gene is not necessary for the photoperiodic response in mice.     

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

Expression of Neuronal Kinesin Heavy Chain Is Developmentally Regulated in the Central Nervous System of the Rat

Abstract: The kinesin family of motor proteins comprises at least two isoforms of conventional kinesin encoded by different genes: ubiquitous kinesin, expressed in all cells and tissues, and neuronal kinesin, expressed exclusively in neuronal cells. In the present study, we have analyzed the expression of the two kinesin isoforms by immunochemistry at different stages of development of the rat CNS. We have found that the level of expression of neuronal kinesin is five to eight times higher in developing than in adult rat brains, whereas that of ubiquitous kinesin is only ∼2.5 times higher in maturing versus adult brains. Moreover, we have studied the distribution of neuronal kinesin by light microscopic immunocytochemistry in the rat brain at different postnatal ages and have found this protein not only to be more highly expressed in juvenile than in adult rat brains but also to show a different pattern of distribution. In particular, tracts of axonal fibers were clearly stained at early postnatal stages of development but were markedly unlabeled in adult rat brains. Our results indicate that the expression of at least one isoform of conventional neuron-specific kinesin is up-regulated in the developing rat CNS and suggest that this protein might play an important role in microtubule-based transport during the maturation of neuronal cells in vivo.     

The Luteinizing Hormone Surge Regulates Circadian Clock Gene Expression in the Chicken Ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)     

Palmdelphin在出生后发育的大鼠小脑中的表达模式研究

Palmdelphin是参与质膜的动态变化与细胞形态的调控的paralemmin家族新成员,与神经发育的相关性尚不明确.前期工作提示,它与调控小脑发育的一种肌动蛋白结合蛋白Mtss1(metastasis suppressor1)具有一定相关性.为了探索该基因与小脑出生后发育的相关性,利用原位杂交技术研究Palmdelphin在小脑中的时空表达,结果表明,Palmdelphin在出生后第7d大鼠小脑中有明显的表达,且分布主要集中在浦肯野神经元.半定量RT-PCR的结果进一步表明Palmdelphin的转录水平在小脑发育过程中受到调控,在出生后7d有表达高峰.这些结果显示Palmdelphin与小脑出生后神经元发育存在一定相关性.