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1.
We have examined spinal motor neurons in Sprague-Dawley rats to further characterize a mechanoenzyme, myosin-Igamma (myr4), which is found in high concentration during axon tract formation in neonates. We raised an antibody to myr4 and made riboprobes for in situ hybridization. Myr4 mRNA was abundant in spinal cord motor neurons (particularly during axon regrowth). Nerves undergoing Wallerian degeneration (from a crush 7 days earlier) showed anti-myr4 labeling of the axolemma and SER--after microtubules, neurofilaments, and F-actin had already been degraded--which is consistent with a described lipid-binding domain in the tail region of myosin-Is. Newly synthesized myr4 was carried in axons by the slow component (SC) of axonal transport at 1-8 mm/day, whereas, none was carried by the fast component (FC). We conclude that SC delivers myr4 to the cytoplasmic surfaces of stationary axonal membranes (SER and axolemma). This positioning would anchor the tail domain of myr4 and leave the catalytic head domain free to interact with F-actin.  相似文献   

2.
《The Journal of cell biology》1993,120(6):1393-1403
We have identified, characterized and cloned a novel mammalian myosin-I motor-molecule, called myr 1 (myosin-I from rat). Myr 1 exists in three alternative splice forms: myr 1a, myr 1b, and myr 1c. These splice forms differ in their numbers of putative calmodulin/light chain binding sites. Myr 1a-c were selectively released by ATP, bound in a nucleotide-dependent manner to F-actin and exhibited amino acid sequences characteristic of myosin-I motor domains. In addition to the motor domain, they contained a regulatory domain with up to six putative calmodulin/light chain binding sites and a tail domain. The tail domain exhibited 47% amino acid sequence identity to the brush border myosin-I tail domain, demonstrating that myr 1 is related to the only other mammalian myosin-I motor molecule that has been characterized so far. In contrast to brush border myosin-I which is expressed in mature enterocytes, myr 1 splice forms were differentially expressed in all tested tissues. Therefore, myr 1 is the first mammalian myosin-I motor molecule with a widespread tissue distribution in neonatal and adult tissues. The myr 1a splice form was preferentially expressed in neuronal tissues. Its expression was developmentally regulated during rat forebrain ontogeny and subcellular fractionation revealed an enrichment in purified growth cone particles, data consistent with a role for myr 1a in neuronal development.  相似文献   

3.
In an effort to determine diversity and function of mammalian myosin I molecules, we report here the cloning and characterization of myr 3 (third unconventional myosin from rat), a novel mammalian myosin I from rat tissues that is related to myosin I molecules from protozoa. Like the protozoan myosin I molecules, myr 3 consists of a myosin head domain, a single light chain binding motif, and a tail region that includes a COOH-terminal SH3 domain. However, myr 3 lacks the regulatory phosphorylation site present in the head domain of protozoan myosin I molecules. Evidence was obtained that the COOH terminus of the tail domain is involved in regulating F-actin binding activity of the NH2-terminal head domain. The light chain of myr 3 was identified as the Ca(2+)-binding protein calmodulin. Northern blot and immunoblot analyses revealed that myr 3 is expressed in many tissues and cell lines. Immunofluorescence studies with anti-myr 3 antibodies in NRK cells demonstrated that myr 3 is localized in the cytoplasm and in elongated structures at regions of cell-cell contact. These elongated structures contained F-actin and alpha-actinin but were devoid of vinculin. Incubation of NRK cells with Con A stimulated the formation of myr 3-containing structures along cell-cell contacts. These results suggest for myr 3 a function mediated by cell-cell contact.  相似文献   

4.
We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I''s. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F- actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.  相似文献   

5.
Efficient control of Shigella -induced, rho-dependent cytoskeletal rearrangements seems to be required to shape the delicate cellular structures associated with bacterial invasion of epithelial cells. We therefore studied a class IX myosin and rho antagonist, the GTPase-activating protein (GAP) myr5, for a potential role in the bacterial entry process. We show that myr5 is recruited into bacterial entry spots. The recruitment pattern resembled that of rhoC or ezrin, but not rhoA, rac or CDC42, while in vitro GAP activity of myr5 was similar for rhoA, B or C. Analysis of myr5 mutants suggested that GTPase- or ATP-binding activites are not required for Shigella -induced recruitment of this atypical myosin to the bacterial entry site. Functional studies revealed a potential dual role of the myosin functions and the GAP module of myr5 for bacterial internalization.  相似文献   

6.
Sequencing of a cytochrome oxidase II (COII) gene fragment in Bacillus taxa provided evidence that the bisexual B. rossius is the maternal ancestor of the hybridogenetic B. rossius-grandii strains and revealed the same ancestry for both parthenogenetic hybrids: the diploid B. whitei (B. rossius/grandii grandii) and the triploid B. lynceorum (B. rossius/grandii grandii/atticus). Present data clearly demonstrate that all Bacillus unisexuals arose through asymmetrical hybridization events and realized a paraphyletic derivation from the B. rossius redtenbacheri subspecies. The invention of B. rossius mitochondrial DNA haplotypes in specimens with B. grandii grandii nuclear genomes revealed the occurrence of androgenesis in nature. Natural androgens represent a peculiar escape from hybridity and can help maintain the hybridogenetic system through the production of the fathering taxon via hybrid females. Results from the COII gene support the phyletic relationships among taxa suggested by previous taxonomical approaches, but also indicate a departure of B. grandii subspecies from the established taxonomy. Assuming the existence of a molecular clock, the evaluated substitution rate brings the splitting between B. rossius and B. grandii/B. atticus back to 22.79 +/- 2.65 myr before present, while the origin of hybrids appears to be much more recent (1.06 +/- 0.53 myr).  相似文献   

7.
Seagrasses are composed of four families belonging to angiosperms and they are thought to become adaptive to aquatic life independently. Zosteraceae is one such family and because of the relatively high species diversity around Japan and Korea coast areas, the family might have arisen therefrom. To elucidate the origin and evolution of Zosteraceae which consists of three genera, Phyllospadix, Zostera, and Heterozostera, 2.8 kb nucleotide sequences of rbcL and matK genes in the chloroplast genome were examined for various species, including cosmopolitan Z. marina and endemic Z. caulescens. The phylogenetic analysis reveals the following three features. First, based on the synonymous nucleotide substitution rate of the rice chloroplast genome, we estimated the divergence times between Zosteraceae and its closest relative, Potamogetonaceae, and between different genera, Zostera and Phyllospadix, as approximately 100 million years (myr) and 36 myr, respectively, suggesting that Zosteraceae emerged somewhere in the period from 36 myr ago to 100 myr ago. Second, two subgenera of Zostera, Zostera and Zosterella, exhibit their reciprocal monophyly and appear to have differentiated from each other approximately 33 myr ago. However, the third genus Heterozostera branched off only 5 myr ago from the stem lineage leading to Zosterella and this seems too recent in comparison with the ancient divergence of the two subgenera. Third, we estimated the most recent common ancestor of subgenus Zostera as 6 myr. In Z. marina four haplotypes were found in the sample and have diversified in the past 1.5 myr. One haplotype is shared by both sides of the Japan Archipelago and its closely related haplotypes occur also in eastern Pacific Ocean. Based on these phylogeographic analyses, we propose a provisional age related classification of Zosteraceae to argue the origin and evolution.  相似文献   

8.
Changes in the duration, quality and intensity of light affect flowering time. Compared with the effects of light duration and quality, less is known about the effects of light intensity on flowering. Here we describe two paralogous single Myb domain genes, MYB‐RELATED PROTEIN 1 (MYR1) and MYB‐RELATED PROTEIN 2 (MYR2), and their roles as repressors of responses to decreased light intensity in Arabidopsis. Homozygous myr1 myr2 double mutants flowered early under low light intensities. Additionally, myr1 myr2 mutants exhibited increases in petiole length, leaf angle and apical dominance. Genetic analyses involving mutants in the long‐day, gibberellin (GA) and phyB flowering pathways indicated that all aspects of the myr1 myr2 phenotype required GA biosynthesis. The early‐flowering phenotype of myr1 myr2 also required FLOWERING LOCUS T, and myr1 myr2 mutants showed an epistatic interaction with the phyB‐9 mutant. Over‐expression of MYR1 or MYR2 produced GA‐deficiency symptoms that were rescued by application of gibberellic acid (GA3). Loss of MYR1 and MYR2 function was associated with a twofold increase in GA20ox2 expression and a 30% increase in GA4 levels, while over‐expression of MYR2 led to a threefold decrease in GA20ox2 expression and a 50% decrease in GA4 levels. Considered together, these results suggest that the ability of MYR1 and MYR2 to repress flowering and organ elongation is at least partly due to their negative effect on levels of bioactive GA.  相似文献   

9.
Neuronal calcium sensor-1 (NCS-1) interacts with many membranes and cytosolic proteins, both in a Ca2+-dependent and in a Ca2+-independent manner, and its physiological role is governed by its N-terminal myristoylation. To understand the role of myristoylation in altering Ca2+ response and other basic biophysical properties, we have characterized the Ca2+ filling pathways in both myristoylated (myr) and non-myristoylated (non-myr) forms of NCS-1. We have observed that Ca2+ binds simultaneously to all three active EF-hands in non-myr NCS-1, whereas in the case of myr NCS-1, the process is sequential, where the second EF-hand is filled first, followed by the third and fourth EF-hands. In the case of myr NCS-1, the observed sequential Ca2+ binding process becomes more prominent in the presence of Mg2+. Besides, the analysis of 15N-relaxation data reveals that non-myr NCS-1 is more dynamic than myr NCS-1. The overall molecular tumbling correlation time increases by approximately 20% upon myristoylation. Comparing the apo forms of non-myr NCS-1 and myr NCS-1, we found the possibility of existence of some substates, which are structurally closer to the holo form of the protein. There are more such substates in the case of non-myr NCS-1 than in the case of the myr NCS-1, suggesting that the former accesses larger volumes of conformational substates compared with the latter. Further, the study reveals that the possibility of Ca2+ binding simultaneously to different parts of the protein is more favourable in non-myr NCS-1 than in myr NCS-1.  相似文献   

10.
Human immunodeficiency virus type 1 (HIV-1) encodes a polypeptide called Gag that is capable of forming virus-like particles (VLPs) in vitro in the absence of other cellular or viral constituents. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. A combination of in vivo, in vitro, and structural studies have shown that Gag targeting and assembly on the PM are mediated by specific interactions between the myristoylated matrix [myr(+)MA] domain of Gag and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Exposure of the MA myristyl (myr) group is triggered by PI(4,5)P2 binding and is enhanced by factors that promote protein self-association. In the studies reported here, we demonstrate that myr exposure in MA is modulated by pH. Our data show that deprotonation of the His89 imidazole ring in myr(+)MA destabilizes the salt bridge formed between His89(Hδ2) and Glu12(COO-), leading to tight sequestration of the myr group and a shift in the equilibrium from trimer to monomer. Furthermore, we show that oligomerization of a Gag-like construct containing matrix-capsid is also pH-dependent. Disruption of the His?Glu salt bridge by single-amino acid substitutions greatly altered the myr-sequestered?myr-exposed equilibrium. In vivo intracellular localization data revealed that the H89G mutation retargets Gag to intracellular compartments and severely inhibits virus production. Our findings reveal that the MA domain acts as a “pH sensor” in vitro, suggesting that the effect of pH on HIV-1 Gag targeting and binding to the PM warrants investigation.  相似文献   

11.
A novel widely expressed type of myosin (fifth unconventional myosin from rat: myr 5) from rat tissues, defining a ninth class of myosins, was identified. The predicted amino acid sequence of myr 5 exhibits several features not found previously in myosins. The myosin head domain contains a unique N-terminal extension and an insertion of 120 amino acids at a postulated myosin-actin contact site. Nevertheless, myr 5 is able to bind actin filaments in an ATP-regulated manner. The head domain is followed by four putative light chain binding sites. The tail domain of myr 5 contains a region which coordinates two atoms of zinc followed by a region that stimulates GTP hydrolysis of members of the ras-related rho subfamily of small G-proteins. Myr 5 therefore provides the first direct link between rho GTPases which have been implicated in the regulation of actin organization and the actin cytoskeleton. It is also the first unconventional myosin for which a tail binding partner(s), namely members of the rho family, has been identified.  相似文献   

12.
Protein kinase B (PKB), a serine threonine kinase is critically involved in cellular proliferation and survival. To characterize its role in T cell development in vivo, we have analyzed transgenic mice that express a membrane-targeted constitutively active version of PKB (myr PKB) in thymocytes and peripheral T cells. We report that myr PKB renders proliferative responses of thymocytes more sensitive to TCR signals by increased and sustained activation of Src kinase Lck and the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In addition, the proliferative response of myr PKB T cells is relatively independent of calcium mobilization and calcineurin activity. We also find that myr PKB enhances phosphorylation of glycogen synthase kinase 3, a negative regulator of NFAT and T cell activation, and the recruitment of the adapter protein Cbl-c. Interestingly, we demonstrate that upon TCR/CD3 stimulation of wild-type T cells PKB is translocated into lipid rafts, adding a new role for PKB in TCR-initiated signalosome formation in T cell activation. Localization of transgenic PKB in lipid rafts could contribute to the higher TCR sensitivity of myr PKB thymocytes which is reflected in an increase in positive selection toward the CD4 lineage and variable effects on negative selection depending on the model system analyzed. Thus, our observations clearly indicate a cross-talk between PKB and important signaling molecules downstream of TCR that modulate the thresholds of thymocyte selection and T cell activation.  相似文献   

13.
A family of functional neogenes called Mart, related to the gag gene of Sushi-like long terminal repeat retrotransposons from fish and amphibians, is present in the genome of human (11 genes) and other primates, as well as in mouse (11 genes), rat, dog (12 genes), cat, and cow. Mart genes have lost their capacity of retrotransposition through non-functionalizing rearrangements having principally affected long terminal repeats and pol open reading frame. Most Mart genes are located on the X chromosome in different mammals. Sequence database analysis suggested that Mart genes are present in opossum (marsupial), but absent from the genome of chicken. Hence, the Mart gene family might have been formed from Sushi-like retrotransposon(s) after the split of birds and mammals (310 myr ago), but before the divergence between placental mammals and marsupials (170 myr ago). RT-PCR analysis showed that at least six Mart genes are expressed during mouse embryonic development, with in situ hybridization analysis revealing rather ubiquitous expression patterns. Mart expression was also detected in adult mice, with some genes being expressed in all tissues tested, while others showed a much more restricted expression pattern. Although additional analysis will be required to establish the function of the retrotransposon-derived Mart neogenes, these observations support the evolutionary importance of retrotransposable elements as a source of genetic novelty.  相似文献   

14.
Steady progress has been made in defining both the viral and cellular determinants of retroviral assembly and release. Although it is widely accepted that targeting of the Gag polypeptide to the plasma membrane is critical for proper assembly of HIV-1, the intracellular interactions and trafficking of Gag to its assembly sites in the infected cell are poorly understood. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM), a ubiquitous and highly conserved Ca(2+)-binding protein expressed in all eukaryotic cells, and is implicated in a variety of cellular functions. Binding of HIV-1 Gag to CaM is dependent on calcium and is mediated by the N-terminally myristoylated matrix (myr(+)MA) domain. Herein, we demonstrate that CaM binds to myr(+)MA with a dissociation constant (K(d)) of ~2 μm and 1:1 stoichiometry. Strikingly, our data revealed that CaM binding to MA induces the extrusion of the myr group. However, in contrast to all known examples of CaM-binding myristoylated proteins, our data show that the myr group is exposed to solvent and not involved in CaM binding. The interactions between CaM and myr(+)MA are endothermic and entropically driven, suggesting that hydrophobic contacts are critical for binding. As revealed by NMR data, both CaM and MA appear to engage substantial regions and/or undergo significant conformational changes upon binding. We believe that our findings will provide new insights on how Gag may interact with CaM during the HIV replication cycle.  相似文献   

15.
Abstract:  The end-Permian mass extinction, 252 million years (myr) ago, marks a major shift in the posture of tetrapods. Before the mass extinction, terrestrial tetrapods were sprawlers, walking with their limbs extended to the sides; after the event, most large tetrapods had adopted an erect posture with their limbs tucked under the body. This shift had been suspected from the study of skeletal fossils, but had been documented as a long process that occupied some 15–20 myr of the Triassic. This study reads posture directly from fossil tracks, using a clear criterion for sprawling vs erect posture. The track record is richer than the skeletal record, especially for the Early and Middle Triassic intervals, the critical 20 myr during which period the postural shift occurred. The shift to erect posture was completed within the 6 myr of the Early Triassic and affected both lineages of medium to large tetrapods of the time, the diapsids and synapsids.  相似文献   

16.
Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.  相似文献   

17.
Abstract: Microvertebrate sampling of the Stairway Sandstone (Darriwilian, Middle Ordovician, central Australia) has yielded scales that are chondrichthyan‐like in their overall construction, and Tantalepis gatehousei gen. et sp. nov. is erected here to describe these specimens. Tantalepis gatehousei gen. et sp. nov. is the stratigraphically oldest microsquamous taxon described thus far, and the ‘shark‐like’ appearance of the scales may extend the chondrichthyan lineage back into the Middle Ordovician. The presence of ‘shark‐like’ scales in the fossil record some 50 myr prior to the first articulated chondrichthyan body fossils and 44 myr before the first clearly identifiable chondrichthyan teeth suggests there is a considerable scope for the recovery of articulated specimens with which to document the early history of crown gnathostomes. Traditional hypotheses of phylogenetic relationships among early jawed vertebrates were recently challenged by the proposal of a radically different tree topology. However, the development of a new data set specifically addressing scale‐based characters is required before taxa such as Tantalepis, that are based upon disarticulated remains alone, can be firmly placed within the emerging, revised, evolutionary narrative.  相似文献   

18.
“蓝田人”的磁性地层年龄   总被引:37,自引:4,他引:33  
蓝田附近两个地点更新世黄土中发现的蓝田人化石具有不同的年代。公王岭发现的蓝田人头盖骨的年代为距今115万年,陈家窝的下颌骨年代为距今65万年。年代资料来自于新的磁性地层学研究和黄土-古土壤序列的对比结果。公王岭地点蓝田人化石埋藏于反映了干冷气候的粉砂质黄土中的事实,以及与其相伴的“南方色彩”的哺乳动物群的存在表明,距今115万年前中国北方可能发生了一次重要的秦岭强烈隆起和气候干冷的地质事件。  相似文献   

19.
Assembly of human T-cell leukemia virus type 1 (HTLV-1) particles is initiated by the trafficking of virally encoded Gag polyproteins to the inner leaflet of the plasma membrane (PM). Gag–PM interactions are mediated by the matrix (MA) domain, which contains a myristoyl group (myr) and a basic patch formed by lysine and arginine residues. For many retroviruses, Gag–PM interactions are mediated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]; however, previous studies suggested that HTLV-1 Gag–PM interactions and therefore virus assembly are less dependent on PI(4,5)P2. We have recently shown that PI(4,5)P2 binds directly to HTLV-1 unmyristoylated MA [myr(–)MA] and that myr(–)MA binding to membranes is significantly enhanced by inclusion of phosphatidylserine (PS) and PI(4,5)P2. Herein, we employed structural, biophysical, biochemical, mutagenesis, and cell-based assays to identify residues involved in MA–membrane interactions. Our data revealed that the lysine-rich motif (Lys47, Lys48, and Lys51) constitutes the primary PI(4,5)P2–binding site. Furthermore, we show that arginine residues 3, 7, 14 and 17 located in the unstructured N-terminus are essential for MA binding to membranes containing PS and/or PI(4,5)P2. Substitution of lysine and arginine residues severely attenuated virus-like particle production, but only the lysine residues could be clearly correlated with reduced PM binding. These results support a mechanism by which HTLV-1 Gag targeting to the PM is mediated by a trio engagement of the myr group, Arg-rich and Lys-rich motifs. These findings advance our understanding of a key step in retroviral particle assembly.  相似文献   

20.
David Penney 《Palaeontology》2004,47(2):367-375
The oldest described fossils of the extant spider family Araneidae (Araneinae; gen. et sp. indet.), the extant genus Orchestina (Oonopidae; O. sp. indet.) and the new fossil genus Palaeosegestria (Segestriidae; P. lutzzii gen. et sp. nov.) are presented from Upper Cretaceous amber of New Jersey. The known fossil range of the extant family Araneidae is extended approximately 50 myr from the previously oldest described araneid from the Middle Eocene oil shales of the Messel pit in Hesse, Germany. The fossil range of the extant genus Orchestina is also extended 50 myr from the previously oldest described specimen in Eocene Baltic amber.  相似文献   

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