FXY2/MID2, a gene related to the X-linked Opitz syndrome gene FXY/MID1, maps to Xq22 and encodes a FNIII domain-containing protein that associates with microtubules

Opitz G/BBB syndrome (OS) is a genetically heterogeneous disorder with an X-linked locus and an autosomal locus linked to 22q11.2. OS affects multiple organ systems with often variable severity even between siblings. The clinical features, which include hypertelorism, cleft lip and palate, defects of cardiac septation, hypospadias, and anorectal anomalies, indicate an underlying disturbance of the developing ventral midline of the embryo. The gene responsible for X-linked OS, FXY/MID1, is located on the short arm of the human X chromosome within Xp22.3 and encodes a protein with both an RBCC (RING finger, B-box, coiled coil) and a B30.2 domain. The Fxy gene in mice is also located on the X chromosome but spans the pseudoautosomal boundary in this species. Here we describe a gene closely related to FXY/MID1, called FXY2, which also maps to the X chromosome within Xq22. The mouse Fxy2 gene is located on the distal part of the mouse X chromosome within a region syntenic to Xq22. Analysis of genes flanking both FXY/MID1 and FXY2 (as well as their counterparts in mouse) suggests that these regions may have arisen as a result of an intrachromosomal duplication on an ancestral X chromosome. We have also identified in both FXY2 and FXY/MID1 proteins a conserved fibronectin type III domain located between the RBCC and B30.2 domains that has implications for understanding protein function. The FXY/MID1 protein has previously been shown to colocalize with microtubules, and here we show that the FXY2 protein similarly associates with microtubules in a manner that is dependent on the carboxy-terminal B30.2 domain.     

MappingTNNC1, the Gene That Encodes Cardiac Troponin I in the Human and the Mouse

We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7.     

The Drosophila Gene abnormal spindle Encodes a Novel Microtubule-associated Protein That Associates with the Polar Regions of the Mitotic Spindle

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp¹ directs the synthesis of a COOH terminally truncated or internally deleted peptide of ~124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH₂-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34^cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.     

PLAC1, an Xq26 Gene with Placenta-Specific Expression

A novel human X-linked gene shows placenta-specific expression and has been named PLAC1. The gene maps 65 kb telomeric to HPRT at Xq26 and has been completely sequenced at the cDNA and genomic levels. The mouse orthologue Plac1 maps to the syntenically equivalent region of the mouse X chromosome. In situ hybridization studies with the antisense mRNA during mouse embryogenesis detect Plac1 expression from 7.5 dpc (days postcoitum) to 14.5 dpc in ectoplacental cone, giant cells, and labyrinthine trophoblasts. The putative human and murine PLAC1 proteins are 60% identical and 77% homologous. Both include a signal peptide and a peptide sequence also found in an interaction domain of the ZP3 (zona pellucida 3) protein. These results make PLAC1 a marker for placental development, with a possible role in the establishment of the mother–fetus interface.     

The Saccharomyces Cerevisiae Spt8 Gene Encodes a Very Acidic Protein That Is Functionally Related to Spt3 and Tata-Binding Protein

Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8 gene and have shown that it encodes a predicted protein of 602 amino acids with an extremely acidic amino terminus. In addition, the predicted SPT8 amino acid sequence contains one copy of a sequence motif found in multiple copies in a number of other eukaryotic proteins, including the β subunit of heterotrimeric G proteins. To investigate further the relationship between SPT8, SPT3 and TBP, we have analyzed the effect of an spt8 null mutation in combination with different spt3 and spt15 mutations. This genetic analysis has shown that an spt8 deletion mutation is suppressed by particular spt3 alleles. Taken together with previous results, these data suggest that the SPT8 protein is required, directly or indirectly, for TBP function at particular promoters and that the role of SPT8 may be to promote a functional interaction between SPT3 and TBP.     

Characterization of a Mouse Gene (Fbxw6) That Encodes a Homologue of Caenorhabditis elegans SEL-10

The SCF complex is a type of ubiquitin ligase that consists of the invariable components SKP1, CUL1, and RBX1 as well as a variable component, known as an F-box protein, that is the main determinant of substrate specificity. The Caenorhabditis elegans F-box- and WD40-repeat-containing protein SEL-10 functionally and physically associates with LIN-12 and SEL-12, orthologues of mammalian Notch and presenilin, respectively. We have now identified a gene (which we call Fbxw6) that encodes a mouse homologue (F-box–WD40 repeat protein 6, or FBW6) of SEL-10 and is expressed mainly in brain, heart, and testis. Co-immunoprecipitation analysis showed that FBW6 interacts with SKP1 and CUL1, indicating that these three proteins form an SCF complex. Comparison of the genomic organization of Fbxw6, which is located on mouse chromosome 3.3E3, with that of mouse Fbxw1, Fbxw2, and Fbxw4 showed only a low level of similarity, indicating that these genes diverged relatively early and thereafter evolved independently.     

Characterization and Developmental Expression of Pax9, a Paired-Box-Containing Gene Related to Pax1

Pax9, a recently identified mouse paired-box-containing gene, is highly homologous to Pax1 and belongs to the same subfamily as Pax1, Hup48, PAX9, and pox meso. Two overlapping cDNA clones spanning the entire coding region of Pax9 were isolated and sequenced. A comparison of the Pax1 and -9 protein sequences reveals a high degree of similarity even outside the paired box, while the carboxy-terminus of the two proteins diverges completely. We demonstrate that Pax9 can bind to the e5 sequence from the Drosophila even skipped promoter, which is also recognized by Pax1. We analyzed the expression of Pax9 during embryo-genesis of wildtype, Undulated short-tail (Un^s), and Danforth's short tail (Sd) mice. In wildtype embryos Pax9 is expressed in the pharyngeal pouches and their derivatives, the developing vertebral column, the tail, the head, and the limbs. Expression of Pax9 is unaffected in Un^s embryos, in which the Pax1 gene is deleted, arguing that expression of Pax9 is not dependent on Pax1. The expression of Pax9 is lost in the caudal part of Sd homozygous embryos, suggesting that expression of Pax9 in the vertebral column independent on the notochord. These results indicate that both Pax9 and -1 may act in parallel during morphogenesis of the vertebral column.     

Cloning and Characterization of a Gene (RFN22) Encoding a Novel Brain Expressed Ring Finger Protein (BERP) That Maps to Human Chromosome 11p15.5

We have recently identified a novel RING finger protein expressed in the rat brain, which associates with myosin V and α-actinin-4. Here we have cloned and characterized the orthologous human BERP cDNA and gene (HGMW-approved symbol RNF22). The human BERP protein is encoded by 11 exons ranging in size from 71 to 733 bp, and fluorescence in situ hybridization shows that the BERP gene maps to chromosome 11p15.5, 3′ to the FE65 gene. The human BERP protein is 98% identical to the rat and mouse proteins, and we have identified a highly conserved potential orthologue in Caenorhabditis elegans. BERP belongs to the RING finger–B-box–coiled coil (RBCC) subgroup of RING finger proteins, and a cluster of these RBCC protein genes is present in chromosome 11p15. Chromosome region 11p15 is thought to harbor tumor suppressor genes, and deletions of this region occur frequently in several types of human cancers. These observations indicate that BERP may be a novel tumor suppressor gene.     

The MID1/PP2A complex: a key to the pathogenesis of Opitz BBB/G syndrome

Opitz BBB/G syndrome is a monogenic disorder that is characterized by malformations of the ventral midline. Investigations into the underlying genetic defects and the pathobiochemistry of this syndrome have already shed light on the mechanisms of both the physiological and the pathological development of the ventral midline, a complicated multistep process. Moreover, these studies have revealed the ubiquitin-dependent regulation of microtubule-associated phosphatase 2A, a central mechanism in many cellular processes. In this review, we summarize recent findings and speculate upon their implications for both medical and general research.     

AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1''s defense function, which is linked to the activation of ETI.     

ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP₂)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP₂-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.Membrane traffic, the transfer of material between membrane-bound compartments, is needed for such diverse cellular processes as secretion, endocytosis, and changes in cell shape that accompany cell growth, division, and migration (reviewed in references 84, 85, and 87). It is mediated by transport vesicles that are formed by budding from a donor membrane. The process of budding is driven by the assembly of a proteinaceous coat. Once the vesicle is formed, the coat must dissociate to permit fusion with an acceptor membrane and the consequent delivery of the vesicle’s contents. These steps are regulated in part by the Arf family of small GTP-binding proteins (reviewed in references 8, 23, 61, and 63). Arfs are highly conserved and are found in eukaryotes ranging from yeast to humans. The mammalian Arf family is divided into several classes based largely on sequence similarity: class I (Arfs 1 through 3), class II (Arfs 4 and 5), class III (Arf6), and the more distantly related Arf-like (Arl) class. By linking GTP binding and hydrolysis to coat assembly and disassembly, Arfs regulate membrane trafficking at a number of sites. Arf1 has been implicated in endoplasmic reticulum-to-Golgi and intra-Golgi transport, endosome-to-endosome fusion, and synaptic vesicle formation (8, 23, 28, 61, 63, 66). Arf6 has been implicated in regulation of membrane traffic between the plasma membrane and a specialized endocytic compartment, and its function has been linked to cytoskeletal reorganization (25, 26, 71, 73, 74). The specific sites of action of the other Arf family members are not known.The hydrolysis of GTP on Arf requires a GTPase-activating protein (GAP) (19, 61). With multiple Arfs and multiple sites of action, the existence of several unique Arf GAPs had been anticipated. A number of activities have been purified or partially purified from mammalian sources, including rat liver (19, 57, 77), rat spleen (21), and bovine brain (79), and two Arf GAP activities from rat liver have been resolved (77). They have similar Arf specificities but differ in their lipid dependencies. One of the Arf GAPs (ArfGAP/ArfGAP1, hereafter referred to as ArfGAP1) which functions in the Golgi is activated by dioleoglycerols (3, 4, 19, 40). ArfGAP1, in common with a yeast Arf GAP, GCS1 (72), contains a zinc finger domain which is required for activity (19). The second Arf GAP (ArfGAP2) is specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidic acid (PA). Based on lipid requirements, ArfGAP2 was speculated to function at the plasma membrane and be regulated independently of ArfGAP1 (77). ArfGAP1 and ArfGAP2 were antigenically distinct and, therefore, likely to be distinct gene products; however, prior to this study, only ArfGAP1 had been cloned (19).Src, a cytoplasmic tyrosine kinase with N-terminal Src homology 3 (SH3) and SH2 domains, transduces signals important for cell growth and cytoskeletal organization (12, 68, 91). A number of studies suggest that Src is also involved in regulating membrane traffic. Src associates primarily with endosomal membranes and in several cell types has been localized to specialized secretory vesicles, including synaptic vesicles (5, 20, 34, 46, 54, 69, 81). Overexpression of Src accelerates endocytosis (95). In addition, Src associates with or phosphorylates several proteins involved in membrane trafficking (5, 31, 43, 65).Here, we report the purification and cloning of a PIP₂-dependent Arf GAP, ASAP1. ASAP1 contains a zinc finger domain similar to that required for GAP activity in ArfGAP1 and GCS1. ASAP1 also contains a number of domains that are likely to be involved in regulation and/or localization: a pleckstrin homology (PH) domain, three ankyrin (ANK) repeats, a proline-rich region with SH3 binding motifs, and an SH3 domain. In addition, ASAP1 was identified independently as a binding protein for Src and was found to be phosphorylated on tyrosine in cells that express activated Src. ASAP1 also associated with the adapter protein c-Crk in vitro. ASAP1 was localized to the cytoplasm and the cell edge likely associated with the plasma membrane. We propose that ASAP1, by binding both Src and PIP₂, could coordinate membrane trafficking with cell growth or actin cytoskeleton remodeling.     

GPC6, a Novel Member of the Glypican Gene Family, Encodes a Product Structurally Related to GPC4 and Is Colocalized withGPC5on Human Chromosome 13

Glypicans are a family of cell surface heparan sulfate proteoglycans that appear to play an important role in cellular growth control and differentiation, as is supported by the observation that mutations in GPC3 are responsible for Simpson-Golabi-Behmel syndrome (SGBS) in humans. Recently it has been shown that the GPC4 gene is tightly clustered with GPC3 on the X chromosome and that some patients with SGBS apparently have deletions affecting both genes. We report here the identification of a human cDNA encoding a novel glypican family member, glypican-6. This cDNA encodes a predicted protein of 554 amino acids and is structurally analogous to other members of the glypican gene family, but most highly related to glypican-4. A single GPC6 mRNA of 6.2 kb is detected most abundantly in the ovary, liver, and kidney, with lower levels of mRNA expression also detected in a wide range of other adult tissues. Radiation hybrid analysis mapped the GPC6 gene to human chromosome 13 very near the GPC5 gene, a member of the glypican family bearing strong similarity to GPC3.     

刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。