检测脱氨基速率的超灵敏方法及应用

脱氨基被认为是引起细胞突变的主要因素,如果这些脱氨基的产物不被修复,将引起转换(transition)突变.为了理解DNA结构和其化学活性的关系,介绍一种新的灵敏的遗传学方法,它应用在DNA特定点的脱氨基速率的测定.这种方法基于M13mp2噬菌体内的1acZα基因中的CCC脯氨酸密码子的反转突变,即每个脱氨基事件表现为在白色菌斑背景中的一个蓝色菌斑,其灵敏度可达10⁵ M13mp2 DNA分子中检验出一个脱氨基事件.此外,该法可以计算脱氨基动力学速率常数和反应活化能.     

A nuclear genetic lesion affecting Saccharomyces cerevisiae mitochondrial translation is complemented by a homologous Bacillus gene.

A novel Bacillus gene was isolated and characterized. It encodes a homolog of Saccharomyces cerevisiae Pet112p, a protein that has no characterized relative and is dispensable for cell viability but required for mitochondrial translation. Expression of the Bacillus protein in yeast, modified to ensure mitochondrial targeting, partially complemented the phenotype of the pet112-1 mutation, demonstrating a high degree of evolutionary conservation for this as yet unidentified component of translation.     

The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.     

Glucose Transporter (GLUT-4) Is Targeted to Secretory Granules in Rat Atrial Cardiomyocytes

The insulin-responsive glucose transporter GLUT-4 is found in muscle and fat cells in the transGolgi reticulum (TGR) and in an intracellular tubulovesicular compartment, from where it undergoes insulindependent movement to the cell surface. To examine the relationship between these GLUT-4–containing compartments and the regulated secretory pathway we have localized GLUT-4 in atrial cardiomyocytes. This cell type secretes an antihypertensive hormone, referred to as the atrial natriuretic factor (ANF), in response to elevated blood pressure. We show that GLUT-4 is targeted in the atrial cell to the TGR and a tubulo-vesicular compartment, which is morphologically and functionally indistinguishable from the intracellular GLUT-4 compartment found in other types of myocytes and in fat cells, and in addition to the ANF secretory granules. Forming ANF granules are present throughout all Golgi cisternae but only become GLUT4 positive in the TGR. The inability of cyclohexamide treatment to effect the TGR localization of GLUT-4 indicates that GLUT-4 enters the ANF secretory granules at the TGR via the recycling pathway and not via the biosynthetic pathway. These data suggest that a large proportion of GLUT-4 must recycle via the TGR in insulin-sensitive cells. It will be important to determine if this is the pathway by which the insulin-regulatable tubulo-vesicular compartment is formed.     

A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.     

ERS-24, a Mammalian v-SNARE Implicated in Vesicle Traffic between the ER and the Golgi

We report the identification and characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE implicated in vesicular transport between the ER and the Golgi. ERS24 is incorporated into 20S docking and fusion particles and disassembles from this complex in an ATP-dependent manner. ERS-24 has significant sequence homology to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the ER and the Golgi. ERS-24 is localized to the ER and to the Golgi, and it is enriched in transport vesicles associated with these organelles.Newly formed transport vesicles have to be selectively targeted to their correct destinations, implying the existence of a set of compartment-specific proteins acting as unique receptor–ligand pairs. Such proteins have now been identified (Söllner et al., 1993a ; Rothman, 1994): one partner efficiently packaged into vesicles, termed a v-SNARE,¹ and the other mainly localized to the target compartment, a t-SNARE. Cognate pairs of v- and t-SNAREs, capable of binding each other specifically, have been identified for the ER–Golgi transport step (Lian and Ferro-Novick, 1993; Søgaard et al., 1994), the Golgi–plasma membrane transport step (Aalto et al., 1993; Protopopov et al., 1993; Brennwald et al., 1994) in Saccharomyces cerevisiae, and regulated exocytosis in neuronal synapses (Söllner et al., 1993a ; for reviews see Scheller, 1995; Südhof, 1995). Additional components, like p115, rab proteins, and sec1 proteins, appear to regulate vesicle docking by controlling the assembly of SNARE complexes (Søgaard et al., 1994; Lian et al., 1994; Sapperstein et al., 1996; Hata et al., 1993; Pevsner et al., 1994).In contrast with vesicle docking, which requires compartment-specific components, the fusion of the two lipid bilayers uses a more general machinery derived, at least in part, from the cytosol (Rothman, 1994), which includes an ATPase, the N-ethylmaleimide–sensitive fusion protein (NSF) (Block et al., 1988; Malhotra et al., 1988), and soluble NSF attachment proteins (SNAPs) (Clary et al., 1990; Clary and Rothman, 1990; Whiteheart et al., 1993). Only the assembled v–t-SNARE complex provides high affinity sites for the consecutive binding of three SNAPs (Söllner et al., 1993b ; Hayashi et al., 1995) and NSF. When NSF is inactivated in vivo, v–t-SNARE complexes accumulate, confirming that NSF is needed for fusion after stable docking (Søgaard et al., 1994).The complex of SNAREs, SNAPs, and NSF can be isolated from detergent extracts of cellular membranes in the presence of ATPγS, or in the presence of ATP but in the absence of Mg²⁺, and sediments at ∼20 Svedberg (20S particle) (Wilson et al., 1992). In the presence of MgATP, the ATPase of NSF disassembles the v–t-SNARE complex and also releases SNAPs. It seems likely that this step somehow initiates fusion.To better understand vesicle flow patterns within cells, it is clearly of interest to identify new SNARE proteins. Presently, the most complete inventory is in yeast, but immunolocalization is difficult in yeast compared with animal cells, and many steps in protein transport have been reconstituted in animal extracts (Rothman, 1992) that have not yet been developed in yeast. Therefore, it is important to create an inventory of SNARE proteins in animal cells. The most unambiguous and direct method for isolating new SNAREs is to exploit their ability to assemble together with SNAPs and NSF into 20S particles and to disassemble into subunits when NSF hydrolyzes ATP. Similar approaches have already been successfully used to isolate new SNAREs implicated in ER to Golgi (Søgaard et al., 1994) and intra-Golgi transport (Nagahama et al., 1996), in addition to the original discovery of SNAREs in the context of neurotransmission (Söllner et al., 1993a ).Using this method, we now report the isolation and detailed characterization of ERS-24 (Endoplasmic Reticulum SNARE of 24 kD), a new mammalian v-SNARE that is localized to the ER and Golgi. ERS-24 is found in transport vesicles associated with the transitional areas of the ER and with the rims of Golgi cisternae, suggesting a role for ERS-24 in vesicular transport between these two compartments.     

Evolutionary Relationship of the Ligand-Gated Ion Channels and the Avermectin-Sensitive,Glutamate-Gated Chloride Channels

Two cDNAs, GluClα and GluClβ, encoding glutamate-gated chloride channel subunits that represent targets of the avermectin class of antiparasitic compounds, have recently been cloned from Caenorhabditis elegans (Cully et al., Nature, 371, 707–711, 1994). Expression studies in Xenopus oocytes showed that GluClα and GluClβ have pharmacological profiles distinct from the glutamate-gated cation channels as well as the γ-aminobutyric acid (GABA)- and glycine-gated chloride channels. Establishing the evolutionary relationship of related proteins can clarify properties and lead to predictions about their structure and function. We have cloned and determined the nucleotide sequence of the GluClα and GluClβ genes. In an attempt to understand the evolutionary relationship of these channels with the members of the ligand-gated ion channel superfamily, we have performed gene structure comparisons and phylogenetic analyses of their nucleotide and predicted amino acid sequences. Gene structure comparisons reveal the presence of several intron positions that are not found in the ligand-gated ion channel superfamily, outlining their distinct evolutionary position. Phylogenetic analyses indicate that GluClα and GluClβ form a monophyletic subbranch in the ligand-gated ion channel superfamily and are related to vertebrate glycine channels/receptors. Glutamate-gated chloride channels, with electrophysiological properties similar to GluClα and GluClβ, have been described in insects and crustaceans, suggesting that the glutamate-gated chloride channel family may be conserved in other invertebrate species. The gene structure and phylogenetic analyses in combination with the distinct pharmacological properties demonstrate that GluClα and GluClβ belong to a discrete ligand-gated ion channel family that may represent genes orthologous to the vertebrate glycine channels. Received: 30 September 1996 / Accepted: 15 November 1996     

Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet-Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.     

Comparison of Type 2 Inositol 1,4,5-Trisphosphate Receptor Distribution and Subcellular Ca2+ Release Sites that Support Ca2+ Waves in Cultured Astrocytes

Abstract: We have examined the mechanisms that underlie Ca²⁺ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca²⁺ waves in astrocytes that began at discrete initiation loci and propagated throughout the cell by regenerative amplification at a number of cellular sites, as shown by very high Ca²⁺ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca²⁺ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5-trisphosphate receptor (InsP₃R) density. To examine this possibility, we compared the distribution pattern of endoplasmic reticulum (ER) and InsP₃Rs with Ca²⁺ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanine iodide staining revealed that the ER in astrocytes exists as a meshwork of membranes extending throughout the cells, including fine processes. A specific antibody directed against type 2 InsP₃Rs (InsP₃R2) detected a 260-kDa band in western blotting of astrocyte membranes. Immunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca²⁺ responses and InsP₃R2 immunofluorescence were compared in the same cell, domains of elevated Ca²⁺ response kinetics (high amplitude and rapid rate of rise) showed significant positive correlation with high local intensity of InsP₃R2 staining. It appears, therefore, that specializations in the ER responsible for discrete local Ca²⁺ release sites that support regenerative wave propagation include increased levels of InsP₃R2 expression.     

Ganglioside GM1 Activates the Mitogen-Activated Protein Kinase Erk2 and p70 S6 Kinase in U-1242 MG Human Glioma Cells

Abstract: Gangliosides are implicated in the regulation of cellular proliferation as evidenced by differences in ganglioside composition associated with malignant transformation and density of cells in culture, as well as their inhibitory effects when added to cells growing in culture. Exogenously added gangliosides have a bimodal effect on proliferation in U-1242 MG glioma cells, inhibiting DNA synthesis in growing cells and stimulating it in quiescent cells. We investigated the mechanisms involved in stimulation of DNA synthesis using [³H]thymidine incorporation and immune complex kinase assays to identify responsible signal transduction pathways. Treatment of quiescent U-1242 MG cells with GM1 caused activation of the mitogen-activated protein (MAP) kinase isoform Erk2. Pretreatment with the specific MAP kinase kinase inhibitor PD98059 prevented the GM1-stimulated Erk2 activation and GM1-stimulated DNA synthesis. GM1 treatment stimulated another distinct signaling pathway leading to activation of p70 S6 kinase (p70^s6k), and this was prevented by pretreatment with rapamycin. Rapamycin also inhibited GM1-stimulated DNA synthesis. Activation of both pathways and stimulation of DNA synthesis were inhibited by forskolin treatment; however, GM1 had no effect on cyclic AMP levels. Platelet-derived growth factor also activated both Erk2 and p70^s6k but did not cause DNA synthesis, suggesting that GM1 may stimulate additional cascades, which also contribute to GM1-mediated DNA synthesis.