Cloning of a Novel Rat Gene, DB83, That Encodes a Putative Membrane Protein

Using a partial cDNA sequence and a 5'-RACE technique, we isolateda novel cDNA from rat liver referred to as DB83. DB83 had fourhydrophobic trans-membrane domains and one N-myristoylationsite as well as multiple possible phosphorylation sites. Thedb83 gene was highly expressed in the liver and significantlyin brain, lungs and kidneys. We suggest that DB83 is a tissue-specificputative membrane protein.     

Coding Sequence, Genomic Organization, and Conserved Chromosomal Localization of the Mouse Gene Scn11a Encoding the Sodium Channel NaN

Previous studies have shown that sodium channel alpha-subunit NaN is preferentially expressed in small-diameter sensory neurons of dorsal root ganglia and trigeminal ganglia. These neurons include high-threshold nociceptors that are involved in transduction of pain associated with tissue and nerve injury. In this study, we show that mouse NaN is a 1765-amino-acid peptide that is predicted to produce a current that is resistant to tetrodotoxin (TTX-R). Mouse and rat NaN are 80 and 89% identical at the nucleotide and amino acid levels, respectively. The Scn11a gene encoding this cDNA is organized into 24 exons. Unlike some alpha-subunits, Scn11a does not have an alternative exon 5 in domain I. Introns of the U2 and U12 spliceosome types are present at conserved positions relative to other members of this family. Scn11a is located on mouse chromosome 9, close to the two other TTX-R sodium channel genes, Scn5a and Scn10a. The human gene, SCN11A, was mapped to the conserved linkage group on chromosome 3p21-p24, close to human SCN5A and SCN10A. The colocalization of the three sodium channel genes supports a common lineage of the TTX-R sodium channels.     

小鼠锌指蛋白基因ZF-12的克隆与基因结构以及5′端启动子活性的分析

人类锌指蛋白ZNF191为类krueppel转录因子,其可能与神经精神病、心血管疾病和肝癌等疾病的发生或发展有关,为了采用基因敲除模型来探讨它的生理功能,克隆和定位其在模式生物（小鼠）中的同源基因,并阐明其结构特征是必需的。通过筛选小鼠λ噬菌体基因组文库,获得了它在小鼠中的同源基因ZF－12基因组全长片段。序列分析表明：该基因含有4个外显子和3个内含子,内含子都遵循GT／AG的剪接模式;第91密码子处存在单核苷酸多态性;可能存在2种大小不同的3′端的非翻译区（3′－UTR）;与锌指蛋白基因Zfp-35相连锁,可将其定位于18号染色体的B3－C带上或附近;5′端上游存在1个约250bp高度富含GC的启动子序列。ZF－12基因的5′端系列缺失荧光素酶基因报告载体的瞬时转染实验表明:其上游序列（-762～ 70bp）具有启动子转录活性,在更上游的序列（－824～-762bp)上可能存在负调控元件。这项研究结果为进一步的基因敲除研究奠定了基础。     

Evidence of Positive Darwinian Selection in Omp85, a Highly ConservedBacterial Outer Membrane Protein Essential for Cell Viability

Omp85 is a highly conserved outer membrane protein found in all gram-negative bacteria. It is essential for bacterial cell viability and plays an integral function in the positioning and folding of other outer membrane proteins into the bacterial outer membrane. We have employed a maximum likelihood and a maximum parsimony approach to detect evidence of positive Darwinian selection in Omp85 homologues from 10 -proteobacteria and have identified 14 amino acid sites that show evidence of being under the influence of adaptive evolution. Interestingly all sites bar one are concentrated within surface loops of the protein that most likely interact with host immune response or the surrounding environment. Alternatively amino acids within membrane-spanning regions of the protein are found to be under purifying selection most likely as a result of structural constraints.Reviewing Editor: Dr. Siv Anderson     

FtsL, an Essential Cytoplasmic Membrane Protein Involved in Cell Division in Escherichia coli

We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome. This gene, which we named ftsL, was detected through characterization of TnphoA insertions in a plasmid containing this chromosomal region. TnphoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus. The ftsL gene is essential for cell growth and division. A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis. Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall. The FtsL protein is estimated to be present at about 20 to 40 copies per cell. The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization.     

BB0250 of Borrelia burgdorferi Is a Conserved and Essential Inner Membrane Protein Required for Cell Division

The gene bb0250 of Borrelia burgdorferi is a homolog of the dedA family, encoding integral inner membrane proteins that are present in nearly all species of bacteria. To date, no precise function has been attributed to any dedA gene. Unlike many bacterial species, such as Escherichia coli, which has eight dedA genes, B. burgdorferi possesses only one, annotated bb0250, providing a unique opportunity to investigate the functions of the dedA family. Here, we show that bb0250 is able to restore normal growth and cell division to a temperature-sensitive E. coli mutant with simultaneous deletions of two dedA genes, yqjA and yghB, and encodes a protein that localizes to the inner membrane of E. coli. The bb0250 gene could be deleted from B. burgdorferi only after introduction of a promoterless bb0250 under the control of an inducible lac promoter, indicating that it is an essential gene in this organism. Growth of the mutant in the absence of isopropyl-β-d-thiogalactopyranoside resulted in cell death, preceded by cell division defects characterized by elongated cells and membrane bulges, demonstrating that bb0250 is required for proper cell division and envelope integrity. Finally, we show that BB0250 depletion leads to imbalanced membrane phospholipid composition in borrelia. These results demonstrate a strong conservation of function of the dedA gene family across diverse species of Gram-negative bacteria and a requirement for this protein family for normal membrane lipid composition and cell division.The dedA family is a highly conserved bacterial gene family encoding inner membrane proteins of unknown function (35). There are more than 2,000 homologs currently found in the NCBI protein database (protein BLAST score versus Escherichia coli DedA of <0.02), and many species of bacteria have multiple homologs. This built-in redundancy has precluded easy genetic analysis. Each of the dedA homologs in E. coli (yqjA, yghB, yabI, yohD, dedA, ydjX, ydjZ, and yqaA) is individually nonessential as the single gene knockouts have been made and are available in the Keio collection (1). Our group has determined that simultaneous deletion of yghB and yqjA from E. coli results in a strain (named BC202; ΔyghB::Kan^r ΔyqjA::Tet^r) that has abnormal membrane phospholipid composition, does not complete cell division (forming chains of cells), and fails to grow at 42°C (35). YghB and YqjA are proteins of 219 and 220 amino acids, respectively, displaying 61% amino acid identity. The other six E. coli homologs display roughly 25 to 30% amino acid identity with each other and YghB/YqjA.The E. coli mutant BC202 referred to above displays several intriguing phenotypes that reflect important functions for the DedA family. The membrane and cell division defects of BC202 are present at both the permissive and nonpermissive growth temperatures. However, BC202 is not hypersensitive to antibiotics or detergents, likely signifying an intact outer membrane, under permissive growth conditions (35). We have demonstrated that the periplasmic amidases AmiA and AmiC are not exported to the periplasm in E. coli mutant BC202 (31). These amidases are normally exported across the inner membrane via the twin arginine transport (Tat) pathway in E. coli (6), a Sec-independent protein export pathway found in many bacteria and also present in archaea and plants (4, 5, 11, 26). AmiA and AmiC are required for normal cell division and envelope integrity (19). ΔTat mutants also display cell division defects due to loss of amidase export (6, 33). Overexpression of the components of the Tat pathway (TatABC) restores normal cell division and growth to BC202 (31). However, BC202 shares some, but not all, phenotypes with ΔTat and amidase mutants. In spite of this progress, a precise function for these genes remains to be determined.We are interested in determining if the functions of dedA family genes are conserved in diverse bacterial species. The spirochete Borrelia burgdorferi is a Gram-negative pathogen that is the cause of Lyme disease (3, 9, 34). B. burgdorferi has a complex enzootic life cycle where it cycles between tick and vertebrate hosts with unique patterns of gene expression to ensure survival in each host (20, 29). The B. burgdorferi genome has been sequenced and consists of one linear chromosome and 21 linear and circular plasmids (17). Notably, its genome possesses only one dedA family homolog, annotated bb0250, present on the linear chromosome. Since tools for the genetic manipulation of B. burgdorferi are available and because of the lack of genome redundancy of dedA genes in this organism, we sought to examine the function and essentiality of B. burgdorferi bb0250. Here, we show that cloned bb0250 can complement the mutant phenotypes of E. coli mutant BC202 and localizes to the inner membrane in E. coli. Furthermore, we have deleted bb0250 from B. burgdorferi, and we demonstrate that it is an essential gene in this organism. Loss of gene expression from an inducible plasmid results in cell division defects, morphological abnormalities, changes in membrane phospholipid composition, and growth arrest, implying a general role for DedA family membrane proteins in cell division and maintenance of proper membrane composition and function. Intriguingly, these phenotypes are independent of any role these proteins may play in the Tat protein export pathway since the B. burgdorferi genome does not encode homologs of TatABC or any proteins with predicted Tat-dependent signal peptides (12). These results demonstrate conserved and important functions for DedA family inner membrane proteins in bacterial cell physiology.     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

油菜叶绿体核糖体蛋白基因rps7的克隆和序列分析(英文)

从甘蓝型油菜 (Brassicanapuscv .H1 65)叶绿体基因组克隆得到了编码核糖体蛋白的基因rps7。经序列分析得知 ,该基因编码区包含 468个核苷酸 ,编码一个分子量为 2 0 1 0 9D、由 1 55个氨基酸组成的蛋白质。该基因的核苷酸和编码的氨基酸序列与烟草对应基因的同源性皆高达 97% ;而与水稻对应基因的同源性分别为 90 %和 84%。该基因不含内含子 ,没有典型的SD序列 ,但在 5’端 - 2 5～- 2 2位发现一个与烟草psbA基因的顺式作用元件RBS2完全相同的TGAT框。与烟草和水稻的同源序列比较 ,该基因在 3’端非编码区变异较大 ,发生了多次插入和缺失。构建了包含该基因在内的一个 1 .0kbDNA的限制性内切酶图谱。所报告的基因序列已登录GenBank。     

The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation,Cell Surface Expression,and Secretion of the Amyloid Precursor Protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases α- and β-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid β peptide (Aβ). At present, little is known about the cellular mechanisms that control APP shedding and Aβ generation. Here, we identified a novel protein, transmembrane protein 59 (TMEM59), as a new modulator of APP shedding. TMEM59 was found to be a ubiquitously expressed, Golgi-localized protein. TMEM59 transfection inhibited complex N- and O-glycosylation of APP in cultured cells. Additionally, TMEM59 induced APP retention in the Golgi and inhibited Aβ generation as well as APP cleavage by α- and β-secretase cleavage, which occur at the plasma membrane and in the endosomes, respectively. Moreover, TMEM59 inhibited the complex N-glycosylation of the prion protein, suggesting a more general modulation of Golgi glycosylation reactions. Importantly, TMEM59 did not affect the secretion of soluble proteins or the α-secretase like shedding of tumor necrosis factor α, demonstrating that TMEM59 did not disturb the general Golgi function. The phenotype of TMEM59 transfection on APP glycosylation and shedding was similar to the one observed in cells lacking conserved oligomeric Golgi (COG) proteins COG1 and COG2. Both proteins are required for normal localization and activity of Golgi glycosylation enzymes. In summary, this study shows that TMEM59 expression modulates complex N- and O-glycosylation and suggests that TMEM59 affects APP shedding by reducing access of APP to the cellular compartments, where it is normally cleaved by α- and β-secretase.     

人细胞生长相关5个新基因的染色体定位及其基因结构

基因的染色体定位对我们研究基因相互关系、基因的组织与进化及理解基因与疾病关系具有重要的意义。本文采用RH-PCR方法及生物信息学方法对PP3898、PP1158、PP753、SP260、HC56等5条人细胞生长相关新基因进行染色体定位,并分析了其基因结构。PP3898及PP1158定位于19p13.3,PP753及SP260定位于1q21.1,HC56定位于17p13.3。PP3898含有19个外显子和18个内含子,可读框为2565bp;PP1158含有7个外显子和6个内含子,可读框为1218bp;SP260含有10个外显子和9个内含子,可读框为690bp;HC56为单外显子,可读框为3141bp。另外,对染色体定位获得的信息进行了分析。 Abstract:Five novel human genes related to cell growth control were newly isolated and identified by high-throughput functional screening.In this paper,the chromosomal localization of these five genes is reported.Radiation hybrid mapping and in silico mapping,and their genomic organization were analyzed respectively.PP3898 and PP1158 were assigned to chromosome 19p13.3,SP260 and PP753 to chromosome 1q21.1,and HC56 to chromosome 17p13.3.PP3898 contains nineteen exons and eighteen introns,PP1158 seven exons and six introns,SP260 ten exons and nine introns,and HC56 only one exon.The implications of chromosomal localization are discussed.     

Molecular Cloning, Sequencing, and Expression of omp-40, the Gene Coding for the Major Outer Membrane Protein from the Acidophilic Bacterium Thiobacillus ferrooxidans

Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane.