The complete genome sequence of Mycoplasma bovis strain Hubei-1

Infection by Mycoplasma bovis (M. bovis) can induce diseases, such as pneumonia and otitis media in young calves and mastitis and arthritis in older animals. Here, we report the finished and annotated genome sequence of M. bovis strain Hubei-1, a strain isolated in 2008 that caused calf pneumonia on a Chinese farm. The genome of M. bovis strain Hubei-1 contains a single circular chromosome of 953,114 bp with a 29.37% GC content. We identified 803 open reading frames (ORFs) that occupy 89.5% of the genome. While 34 ORFs were Hubei-1 specific, 662 ORFs had orthologs in the M. bovis type strain PG45 genome. Genome analysis validated lateral gene transfer between M. bovis and the Mycoplasma mycoides subspecies mycoides, while phylogenetic analysis found that the closest M. bovis neighbor is Mycoplasma agalactiae. Glycerol may be the main carbon and energy source of M. bovis, and most of the biosynthesis pathways were incomplete. We report that 47 lipoproteins, 12 extracellular proteins and 18 transmembrane proteins are phase-variable and may help M. bovis escape the immune response. Besides lipoproteins and phase-variable proteins, genomic analysis found two possible pathogenicity islands, which consist of four genes and 11 genes each, and several other virulence factors including hemolysin, lipoate protein ligase, dihydrolipoamide dehydrogenase, extracellular cysteine protease and 5'-nucleotidase.     

Molecular Cloning and Characterization of a Novel Retinoblastoma-Binding Protein

We describe the isolation and characterization of a novel cDNA encoding a polypeptide that interacts in a yeast two-hybrid system as well as in mammalian cells with the retinoblastoma (RB) protein. This new protein, which we call Rim, consists of 897 amino acids, has two leucine zipper motifs, and has a LECEE sequence previously identified as an RB-binding domain. Rim also has an E1A/CtBP-binding motif and four putative nuclear localization signals.RimmRNA is expressed ubiquitously at low levels in all human adult tissues tested and at much higher levels in several tumor cell lines. TheRimgene (HGMW-approved symbol RBBP8) is localized on human chromosome 18q11.2.     

小鼠新分泌蛋白基因mBolA1的克隆与鉴定

采用生物信息学工具预测与实验相结合的方法得到了一个新的小鼠分泌蛋白基因mBolA1。该基因定位于染色体3F2,cDNA全长为730bp,编码137个氨基酸的蛋白,该蛋白含有一个保守的BolA结构域,等电点为9.05。用RT-PCR方法从鼠的混合cDNA库中克隆到mBolA1。Western blot实验表明mBolA1能从瞬转的COS 7细胞中分泌到细胞培养液中。亚细胞定位显示mBolA1定位于细胞浆,且与高尔基体不共定位,提示它是个非经典分泌途径的分泌蛋白。RT PCR显示mBolA1在组织中广泛表达。它的具体功能有待进一步研究。     

Characterization of a lympho-inhibitory peptide produced by Mycoplasma bovis

Mycoplasma bovis is able to inhibit the mitogen-induced proliferation of bovine lymphocytes. Herein is described the isolation of an immuno-inhibitory peptide from M. bovis. Using size exclusion chromatography, three lympho-suppressive fractions were isolated from M. bovis free supernatant. MALDI-TOF analysis revealed a common peak throughout the suppressive fractions. The purest of these fractions was subjected to N-terminal sequencing, revealing an 84% homologous match with the C-terminus of the M. bovis surface protein VspL (variable surface protein-L). A recombinant of the 26 amino acid peptide was also able to suppress Concanavalin A (ConA)-induced proliferation of bovine lymphocytes. This describes a unique immunosuppressive peptide produced by the bovine respiratory pathogen, M. bovis.     

肺炎支原体重组蛋白抗原的初步鉴定

克隆并表达肺炎支原体(Mycoplasma pneumoniae,Mp)P1黏附蛋白D-2区基因片段,进而对重组蛋白的抗原特异性进行鉴定。应用PCR技术获取目的基因片段,并构建含有目的基因片段的重组质粒,用重组质粒酶切图谱法、PCR扩增及核苷酸测序方法鉴定重组质粒。而后将其转入大肠杆菌BL21菌株,用IPTG诱导目的基因表达,用SDS-PAGE分析重组蛋白的相对分子量,免疫印迹实验鉴定其免疫反应性,并用ELISA实验测定重组蛋白抗原的特异性。结果重组质粒中的p1基因片段经测序后,与GenBank中p1基因核苷酸序列比较,其同源性为99.66%~100%;经SDS-PAGE分析,重组蛋白的相对分子量约为59 ku;免疫印迹实验和ELISA实验证实,Mp免疫血清和Mp感染患者血清都能与重组蛋白发生特异反应。研究中的含P1黏附因子D-2区基因的重组质粒已成功构建,其表达的重组蛋白具有特异的免疫反应性,初步ELISA实验证实,本研究获得的重组蛋白可用于临床标本检测。     

Molecular Characterization of a New Babesia bovis Thrombospondin-Related Anonymous Protein (BbTRAP2)

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.     

Molecular Architecture of a Complex between an Adhesion Protein from the Malaria Parasite and Intracellular Adhesion Molecule 1

The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion.     

Characterization of a Strain of Mycoplasma hominis Lacking 120 kDa Membrane Protein Isolated from Vero Cell Culture

A strain of Mycoplasma hominis lacking a major membrane protein of 120 kDa was isolated from a Vero cell culture. This strain showed very slow growth rate and formed nipple-less colonies on agar medium.     

一个小麦丝氨酸—苏氨酸蛋白激酶基因的克隆和分析

用mRNA差异显示技术在含有抗白粉病基因Pm2 1的小麦 (TriticumaestivumL .)_簇毛麦 (Haynaldiavillosa)6VS/ 6AL易位系 92R137中分离与抗白粉病相关的基因 ,获得一个命名为TaPK1的全长cDNA克隆。序列分析表明 ,它与大豆 (Glycinemax (L .)Merr.)蛋白激酶基因GmPK6高度同源。经推测 ,TaPK1编码 416个氨基酸的多肽 ,属丝氨酸_苏氨酸蛋白激酶家族 ,并具酪氨酸激酶特性。TaPK1是从小麦中分离的新基因。     

一个小麦丝氨酸-苏氨酸蛋白激酶基因的克隆和分析

用mRNA差异显示技术在含有抗白粉病基因Pm21的小麦(Tri ticum aestivum L.) -簇毛麦(Haynaldia villosa) 6VS /6AL易位系92R137中分离与抗白粉病相关的基因,获得一个命名为TaPK1的全长cDNA克隆.序列分析表明,它与大豆(Glycine max (L.) Merr.)蛋白激酶基因GmPK6高度同源.经推测,TaPK1 编码416个氨基酸的多肽,属丝氨酸-苏氨酸蛋白激酶家族,并具酪氨酸激酶特性.TaPK1是从小麦中分离的新基因.     

一条KRAB型锌指蛋白全长新基因的克隆与功能初探

从人胎脑cDNA文库中克隆到一条全长的锌指蛋白新基因的cDNA,命名为ZNF303。序列分析表明,ZNF303的C末端含有7个保守的锌指基序,N末端含有一个KRAB(Krüppel?associated box)结构域。利用肝癌组织表达谱基因芯片杂交证明,该基因在肝癌组织中的表达量有明显降低。认为这种降低可能跟肝癌的形成和转移有密切的关系。利用辐射杂交基因定位技术,得出该基因在人类染色体上的位置是19q13.2。 Abstract: We have cloned a full?length novel zinc finger cDNA of the Krüppel family from human fetal brain cDNA library. Sequence analysis indicates that ZNF303 contains 7 highly conserved zinc finger motifs at the C-terminus and a KRAB(Krüppel-associated box)domain at the N-terminus of the deduced rotein.Hybridization using gene chip of hepatic cancer tissue demonstrates the expressive amount in hepatic cancer tissue is lower than control. We hypothecate that the decreasing of expression amount is related to the formation and metastasis of hepatic cancer.Finally,we show that ZNF303 maps on human chromosome 19q13.2 by radiation hybrid.     

密码子优化的肺炎支原体P1蛋白在大肠杆菌中的克隆表达

目的:获得密码子优化的肺炎支原体P1黏附蛋白优势表位抗原基因,并在大肠杆菌中表达,为临床诊断试剂和疫苗研制打下基础。方法:采用生物信息学分析肺炎支原体P1蛋白的抗原表位,筛选特异性P1蛋白优势表位区;采用大肠杆菌优势密码子,设计上述P1蛋白优势表位基因序列;采用退火PCR技术合成上述基因,并利用载体pGEX-4T-2实现P1优势表位抗原在大肠杆菌中的表达;采用ELISA法对纯化的P1抗原活性进行测定。结果:肺炎支原体P1蛋白特异性抗原表位主要位于1154~1521 aa,获得的P1优化密码子基因平行突变37个稀有密码子和2个终止密码子;在大肠杆菌中表达的GST-P1融合蛋白的相对分子质量为65.9×103,纯化后重组抗原能与肺炎支原体感染者血清发生特异性的免疫反应。结论:采用密码子优化基因合成技术实现了肺炎支原体P1优势表位抗原在大肠杆菌中的高效表达,为肺炎支原体感染的诊断试剂研究提供了重要参考。     

Molecular Cloning,Expression, and Characterization of a Ca2+-Dependent,Membrane-Associated Nuclease of Mycoplasma genitalium

In this study, we identified and characterized the enzymatic properties of MG_186, a calcium-dependent Mycoplasma genitalium nuclease. MG_186 displays the hallmarks of nucleases, as indicated by its amino acid sequence similarity to other nucleases. We cloned, UGA corrected, expressed, purified, and demonstrated that recombinant MG_186 (rMG_186) exhibits nuclease activity similar to that of typical sugar-nonspecific endonucleases and exonucleases. Biochemical characterization indicated that Ca²⁺ alone enhances its activity, which was inhibited by divalent cations, such as Zn²⁺ and Mn²⁺. Chelating agents EGTA and EDTA also inhibited nuclease activity. Mycoplasma membrane fractionation and Triton X-114 phase separation showed that MG_186 was a membrane-associated lipoprotein, and electron microscopy revealed its surface membrane location. Incubation of purified human endometrial cell nuclei with rMG_186 resulted in DNA degradation and morphological changes typical of apoptosis. Further, immunofluorescence analysis of rMG_186-treated nuclei indicated that morphological changes were linked to the disintegration of lamin and the internalization of rMG_186. Since M. genitalium has the capacity to invade eukaryotic cells and localize to the perinuclear and nuclear region of parasitized target cells, MG_186 has the potential to provide M. genitalium, which possesses the smallest genome of any self-replicating cell, with the ability to degrade host nucleic acids both as a source of nucleotide precursors for growth and for pathogenic purposes.Mycoplasma genitalium was first identified as a urogenital tract pathogen in men and subsequently implicated in a range of women pathologies, including pelvic inflammatory diseases, cervicitis, endometritis, salpingitis, and tubal factor infertility (5, 37, 40). In addition to its urogenital niche, M. genitalium has been detected in synovial and respiratory tract specimens (3, 39). M. genitalium DNA sequencing revealed a reduced genome size of 580 kb and a low GC content, along with 482 protein-encoding genes, of which 76 were categorized as hypothetical proteins (18). The streamlined genome of M. genitalium results in gene deficits that dramatically limit its biosynthetic capabilities, leading to a complete dependence on the host for metabolic precursors, such as nucleotides, amino acids, fatty acids, and sterols.Since M. genitalium, like most mollicutes, is unable to synthesize de novo purine and pyrimidine bases (27), it must scavenge nucleotides from the host in order to replicate and persist. Only Mycoplasma penetrans has an orotate-related pathway for converting carbamoyl-phosphate to uridine-5′-monophosphate (34). The importance of nucleases in the life cycle of mycoplasmas is reinforced by their detection in at least 20 Mycoplasma species (26). Purification of membrane-associated Ca²⁺/Mg²⁺-dependent M. penetrans and Mycoplasma hyorhinis nucleases and their relation to mycoplasma survival and pathogenesis have been reported (7, 8, 29, 30). Also, a membrane nuclease gene, mnuA, was identified and cloned from Mycoplasma pulmonis (20, 25). mnuA orthologous sequences were found in M. penetrans, Mycoplasma pneumoniae, Mycoplasma hyopneumoniae, Mycoplasma gallisepticum, and Ureaplasma urealyticum but not in M. genitalium. However, recent nuclease studies with M. hyopneumoniae (nuclease gene designated mhp379) revealed the existence of orthologous sequences in M. genitalium as well as in M. pneumoniae, M. pulmonis, M. gallisepticum, and Mycoplasma synoviae (35).M. genitalium was initially described as an extracellular pathogen. Subsequently, we reported that M. genitalium can be observed in the cytoplasmic and perinuclear regions of infected mammalian cells and can persist long-term within these compartments (4, 13, 24). The latter supports the contention that M. genitalium is capable of intracellular replication and survival. Furthermore, our recent evidence suggests that M. genitalium and its protein products are capable of intranuclear localization within infected endometrial cells (41). Therefore, understanding how M. genitalium overcomes its biosynthetic deficiencies and successfully parasitizes host tissues may provide insights into its biological uniqueness as the smallest pathogen capable of “independent” growth. In this report, we characterized a putative lipoprotein, MG_186, that retains the thermostable nuclease motif found in other bacterial nucleases. The gene encoding MG_186 was cloned and expressed in Escherichia coli, and the biochemical properties of purified recombinant MG_186 (rMG_186) nuclease protein were examined along with its impact on intact nuclei isolated from endometrial cells.     

Cloning, Characterization, and Molecular Application of a Beta-Agarase Gene from Vibrio sp. Strain V134

V134, a marine isolate of the Vibrio genus, was found to produce a new beta-agarase of the GH16 family. The relevant agarase gene agaV was cloned from V134 and conditionally expressed in Escherichia coli. Enzyme activity analysis revealed that the optimum temperature and pH for the purified recombinant agarase were around 40°C and 7.0. AgaV was demonstrated to be useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both gram-positive and gram-negative bacteria.     

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

Background

Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/Principal Findings

Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/Significance

The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.     

Molecular Cloning and Characterization of the PsbP Domain Protein 1 Gene (GhPPD1) in Cotton (Gossypium hirsutum L.)

As a member of the PsbP superfamily in plants, the PsbP protein is essential for the water splitting reaction in photosynthesis. PsbP-like proteins (PPLs) are required for the repair of photodamaged photosystem II (PSII) under high-intensity light and the accumulation of chloroplast NADPH dehydrogenase (NDH). However, to date, studies on PsbP domain proteins (PPDs) are rather limited with literature reported on only PPD5 and PPD6. Other PPDs have not been studied yet. In this study, a cotton PsbP domain protein 1 gene (GhPPD1, GenBank accession no. HQ404251) encoding the PPD1 protein was cloned by rapid amplification of complementary DNA (cDNA) ends-polymerase chain reaction. The full-length cDNA is 951 bp and encodes an open reading frame of 257 amino acids with deduced molecular weight of 29 kDa. The GhPPD1 protein shared 77–86 % identity at the amino acid level with PPD1 isolated from Populus trichocarpa, Ricinus communis, Vitis vinifera, Glycine max, and Arabidopsis thaliana. The corresponding genomic DNA containing three exons and two introns was isolated and analyzed. The 5′ flanking region was also analyzed to identify a group of putative cis-acting elements. DNA gel blot analysis revealed that the GhPPD1 gene has two to five copies in the cotton genome. Additionally, real-time PCR analysis revealed that GhPPD1 was expressed in cotton leaves, anthers, and stems, and its expression level in leaves was much higher than those in anthers and stems. It was minimally expressed in other tissues. On the basis of these results, we propose that GhPPD1 is not only associated with photosynthesis but also related to pollen activity and male fertility.     

Molecular Cloning,Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10⁻¹² cm³/s or 4.17 (±1.38)×10¹⁰ H₂O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.     

Molecular Cloning and Characterization of Protein Kinase C from the Sea Urchin Lytechinus pictus

Protein kinase C (PKC) has been shown to play a role in events involved in fertilization such as activation of the Na⁺/H⁺antiporter and an NADPH dependent oxidase. In addition, it is involved in cell fate programming later in development of the sea urchin embryo. In order to further address the role of PKC in sea urchin development, we have screened a Lytechinus pictus ovary tissue cDNA library and identified one clone for sea urchin protein kinase C (suPKC1). This clone encodes a deduced protein with a molecular mass of 72.4 kDa, which shows strong homology to invertebrate and mammalian protein kinase C (PKC) sequences. PKC has been partially purified from eggs of L. pictus. This kinase activity has been shown to be dependent upon phosphatidylserine, diacylglycerol and Ca²⁺. In agreement with this biochemical data, suPKC1 has a C2 or Ca²⁺-binding domain suggesting its activity would be Ca²⁺-dependent. Polyclonal antibodies raised against peptides of the suPKC1 sequence recognize an antigen of approximately 71 kDa in DE52 fractions that contain PKC activity; this reactivity is not observed in fractions that lack PKC activity. Using a ribonuclease protection assay, we have demonstrated the presence of suPKC1 message throughout developmental stages of the sea urchin embryo.     

Molecular Cloning and Characterization of a cDNA for the {alpha} Subunit of a G Protein from Rice

We report the isolation of a cDNA for the     

Molecular Cloning and Characterization of Galactosylceramide Expression Factor-1 (GEF-1)

A rat brain cDNA clone has been isolated using a eukaryotic cell transient expression system with anti-galactosylceramide (GalCer) monoclonal antibody (MAb), that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), but no homology to UDP-galactose: ceramide galactosyltransferase. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes and cell growth suppression. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of CGT increased significantly in MDCK/GEF-1 cells compared with control cells. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer and sulfatide expression in the epithelial cells as well as in the brain.