Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.     

Maxicircle (mitochondrial) genome sequence (partial) of Leishmania major: gene content, arrangement and composition compared with Leishmania tarentolae

We report 8420 bp of DNA sequence data from the maxicircle (mitochondrial) genome of Leishmania major (MHOM/SU/73/5ASKH), a much larger portion of this genome than has been reported previously from any Leishmania species infecting humans. This region contains 10 partial and complete genes: 5 protein-encoding genes (COII, COIII, ND1, ND7 and Cyt b); two ribosomal RNA subunits (12S and 9S) and three unidentified open reading frames (MURF1, MURF4 (ATPase6) and MURF5), as in the lizard-infecting species L. tarentolae. The genes from L. major exhibit 85-87% identity with those of L. tarentolae at the nucleotide level and 71-94% identity at the amino acid level. Most differences between sequences from the two species are transversions. The gene order and arrangement within the maxicircle of L. major are similar to those in L. tarentolae, but base composition and codon usage differ between the species. Codons assigned for initiation for protein-coding genes available for comparison are similar in five genes in the two species. Pre-editing was identified in some of the protein-coding genes. Short intergenic non-coding regions are also present in L. major as they are in L. tarentolae. Intergenic regions between 9S rRNA and MURF5, MURF1 and ND1 genes are G+C rich and considered to be extensive RNA editing regions. The RNA editing process is likely to be conserved in similar pattern in L. major as in L. tarentolae.     

Artificial exon shuffling between tissue-type plasminogen activator (t-PA) and urokinase (u-PA): a comparative study on the fibrinolytic properties of t-PA/u-PA hybrid proteins

We constructed two human tissue-type plasminogen activator/urokinase (t-PA/u-PA) hybrid cDNAs which were expressed by transfection of mouse Ltk- cells. The properties of the secreted proteins were compared with those of recombinant t-PA (rt-PA) and high molecular weight (HMW) u-PA. The hybrid proteins each contain the amino-terminal fibrin-binding chain of t-PA fused to the carboxy-terminal serine protease moiety of u-PA but differ by a stretch of 13 amino acid residues between kringle 2 of t-PA and the plasmin cleavage site of u-PA. Hybrid protein rt-PA/u-PA I contains amino acids 1-262 of t-PA connected with amino acids 147-411 of u-PA, whereas hybrid protein rt-PA/u-PA II consists of the same t-PA segment and residues 134-411 of u-PA. We demonstrated fibrin binding for rt-PA, whereas the hybrid proteins bind to a lesser extent and HMW u-PA has no affinity for fibrin. Plasminogen activation by either one of the hybrid proteins in the absence of a fibrin substitute was similar to that by HMW u-PA, while rt-PA was much less active. The catalytic efficiency, in the presence of a fibrin substitute, increases more than 2000-fold for rt-PA, about 250-fold for hybrid proteins I and II, and 12-fold for HMW u-PA, respectively. Under these conditions the hybrid proteins are more efficient plasminogen activators than the parental ones. The hybrid molecules form a 1:1 molar complex with the human endothelial plasminogen activator inhibitor (PAI-1), analogous to that formed by rt-PA and HMW u-PA. The relative affinity of rt-PA for PAI-1 is 4.6-fold higher than that of HMW u-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amplification of a new region of DNA in an unselected laboratory stock of L. tarentolae: the T region

A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae. This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania, or the maxicircle of L. tarentolae, nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae.     

Structure and function of human tissue-type plasminogen activator (t-PA)

Full-length tissue-type plasminogen activator (t-PA) cDNA served to construct deletion mutants within the N-terminal "heavy" (H)-chain of the t-PA molecule. The H-chain cDNA consists of an array of structural domains homologous to domains present on other plasma proteins ("finger," "epidermal growth factor," "kringles"). These structural domains have been located on an exon or a set of exons. The endpoints of the deletions nearly coincide with exon-intron junctions of the chromosomal t-PA gene. Recombinant t-PA deletion mutant proteins were obtained after transient expression in mouse Ltk- cells, transfected with SV40-pBR322-derived t-PA cDNA plasmids. It is demonstrated that the serine protease moiety of t-PA and its substrate specificity for plasminogen is entirely contained within the C-terminal "light" (L)-chain of the protein. The presence of cDNA, encoding the t-PA signal peptide preceding the remaining portion of t-PA, suffices to achieve secretion of (mutant) t-PA into the medium. The stimulatory effect of fibrin on the plasminogen activator activity of t-PA was shown to be mediated by the kringle K2 domain and, to a lesser extent, by the finger domain. The other domains on the H-chain, kringle K1, and the epidermal growth-factor-like domain, do not contribute to this property of t-PA. These findings correlate well with the fibrin-binding properties of the rt-PA deletion-mutant proteins, indicating that stimulation of the activity is based on aligning of the substrate plasminogen and its enzyme t-PA on the fibrin matrix. The primary target for endothelial plasminogen activator inhibitor (PAI) is located within the L-chain of t-PA. Deleting specific segments of t-PA H-chain cDNA and subsequent transient expression in mouse Ltk- cells of t-PA deletion-mutant proteins did not affect the formation of a stable complex between mutant t-PA and PAI.     

Cloning and expression of human IFN-γ in <Emphasis Type="Italic">Leishmania tarentolae</Emphasis>

Increasing therapeutic applications for recombinant human interferon-gamma (rhIFN-γ), an antiviral pro-inflammatory cytokine, has broadened interest in optimizing methods for its production. We herein describe a unicellular eukaryotic system, Leishmania tarentolae, a Trypanosomatidae protozoan parasite of gecko Tarentola annularis, which has recently been introduced as a candidate for heterologous gene expression. In this study, the hIFN-γ cDNA was amplified from phyto-hemagglutinin-stimulated peripheral blood mononuclear cells of a healthy blood donor using RT–PCR. In order to express, the rhIFN-γ protein, the resulting cDNA was cloned in two expression cassettes (each containing one copy of hIFN-γ cDNA) and integrated into the small subunit of ribosomal RNA gene of L. tarentolae genome by electroporation. Transformed clones were selected in the presence of appropriate antibiotics. Western blotting of rhIFN-γ and ELISA confirmed the expression and production of 9.5 mg of rhIFN-γ protein/l respectively.     

Amplification of a New Region of DNA in an Unselected Laboratory Stock of L. tarentolae: The T Region

A new DNA amplification is described from an isolate of the lizard parasite Leishmania tarentolae . This DNA is present in up to 50 copies in the Trager line of this species and present but not amplified in all other lines tested. This amplification has been named the T amplification (for Tarentolae/Trager). Restriction enzyme digestion and electrophoresis of total DNA reveal amplified fragments totalling 19 kb following staining with ethidium bromide, a finding confirmed by the use of specific hybridization probes. Much of the amplified T DNA occurs as extra-chromosomal circular molecules. No cross-hybridization was observed between the T region and other amplified DNA of Leishmania , or the maxicircle of L. tarentolae , nor was resistance to methotrexate, chloroquine or primaquine detected in the T-amplified line. Combined with our previous results showing H region amplification in 2 other unselected lab stocks, these data demonstrate the prevalence of apparently spontaneous gene amplifications in L. tarentolae .     

Functional splice sites in a zebrafish LINE and their influence on zebrafish gene expression

Long interspersed elements (LINEs) are transposable elements that exist in many kinds of eukaryotic genomes, where they have a large effect on genome evolution. There are several thousands to hundreds of thousands of LINE copies in each eukaryotic genome. LINE elements are amplified by a mechanism called retrotransposition, in which a LINE-encoded protein reverse transcribes (copies) its own RNA. We previously isolated two retrotransposition-competent LINEs, ZfL2-1 and ZfL2-2, from zebrafish. Although it has generally been thought that LINEs do not have ‘introns’ (because the LINE RNA is used as the template during retrotransposition), we now show that these two LINEs contain multiple putative functional splice sites. We further show that at least one pair of these splice sites is actually functional in zebrafish cells. Moreover, some of these splice sites are coupled with the splicing signal of a host endogenous gene, thereby generating a new chimeric spliced mRNA variant for this gene. Our results suggest the possible role of these LINE splice sites in modulating retrotransposition and host gene expression.     

Similar Gene Estimates from Circular and Linear Standards in Quantitative PCR Analyses Using the Prokaryotic 16S rRNA Gene as a Model

Quantitative PCR (qPCR) is one of the most widely used tools for quantifying absolute numbers of microbial gene copies in test samples. A recent publication showed that circular plasmid DNA standards grossly overestimated numbers of a target gene by as much as 8-fold in a eukaryotic system using quantitative PCR (qPCR) analysis. Overestimation of microbial numbers is a serious concern in industrial settings where qPCR estimates form the basis for quality control or mitigation decisions. Unlike eukaryotes, bacteria and archaea most commonly have circular genomes and plasmids and therefore may not be subject to the same levels of overestimation. Therefore, the feasibility of using circular DNA plasmids as standards for 16S rRNA gene estimates was assayed using these two prokaryotic systems, with the practical advantage being rapid standard preparation for ongoing qPCR analyses. Full-length 16S rRNA gene sequences from Thermovirga lienii and Archaeoglobus fulgidus were cloned and used to generate standards for bacterial and archaeal qPCR reactions, respectively. Estimates of 16S rRNA gene copies were made based on circular and linearized DNA conformations using two genomes from each domain: Desulfovibrio vulgaris, Pseudomonas aeruginosa, Archaeoglobus fulgidus, and Methanocaldocococcus jannaschii. The ratio of estimated to predicted 16S rRNA gene copies ranged from 0.5 to 2.2-fold in bacterial systems and 0.5 to 1.0-fold in archaeal systems, demonstrating that circular plasmid standards did not lead to the gross over-estimates previously reported for eukaryotic systems.     

N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line

To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.     

Efficiency of erythropoietin's signal peptide for HIV(MN)-1 gp 120 expression

The HIV-1 gp120 gene with natural signal sequence expressed in eukaryotic expression systems showed extremely low levels of synthesis and secretion. Several expression systems have been used to improve the secretion levels of gp 120. In mammalian cells, the efficient expression of gp120 fused to t-PA signal peptide has been previously reported. Here, the effects of t-PA and EPO signal peptides were compared as secretion sequences for expression of gp120 in COS-7 cells. The EPO's signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results indicated that higher amounts of secreted gp 120 were obtained when vectors containing EPO signal peptide were used.     

结核分枝杆菌Ag85B-Esat6-HspX融合基因真核表达载体的构建

目的构建结核分枝杆菌Ag85B-Esat6-HspX融合基因,并对其在体外真核细胞中进行表达。方法用PCR法从结核分枝杆菌H37Rv株基因组中分别扩增Ag85B、Esat6、HspX基因,插入到pUC19-T载体,序列测定正确后,将融合基因再次克隆到真核表达载体pcDNA3.1（-）。重组质粒经酶切鉴定并测序正确后,用MegaTran1.0转染293T细胞,并用Western-Blot检测目的蛋白的表达。结果 Western-blot检测到分子量大小约65 kDa的目的蛋白。结论成功地构建了结核分枝杆菌Ag85B-Esat6-HspX融合基因的真核表达载体,且该重组载体可在体外真核细胞中获得特异性的表达。     

Binding of tissue-type plasminogen activator with human endothelial cell monolayers. Characterization of the high affinity interaction with plasminogen activator inhibitor-1

The formation and release of covalent complexes between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) limits the application of equilibrium radioligand binding analysis to characterize the interaction between t-PA and human umbilical vein endothelial cell (HUVEC) monolayers. To avoid this difficulty, we used a recombinant mutant of t-PA, S478A rt-PA, in which alanine has been substituted for the active-site serine. Although the mutant is incapable of covalently reacting with PAI-1, 125I-labeled S478A rt-PA binding to HUVEC monolayers is specific and reversible and is characterized by a high affinity (Kd of 1.5 nM) and a large number of sites (1.5 x 10(6)/cell). This binding was shown to occur through noncovalent interaction with PAI-1 in the HUVEC monolayer by the fact that a monoclonal anti-PAI-1 antibody (MA-7D4) completely blocked S478A rt-PA binding. Two solution-phase assays with recombinant PAI-1 (rPAI-1) confirmed this noncovalent interaction: complexes between 125I-S478A rt-PA and rPAI-1 could be isolated by immunoprecipitation with anti-PAI-1 antibodies, and S478A rt-PA competed with rt-PA for inactivation by rPAI-1. In contrast diisopropylphosphate rt-PA (in which the active site serine is chemically modified) showed minimal binding to HUVEC monolayers, as a result of impaired interaction with PAI-1, in the two assays. Thus, both wild-type rt-PA and S478A rt-PA interact with the HUVEC monolayer through PAI-1. With rt-PA this results in the formation of covalent rt-PA.PAI-1 complexes that are released from the monolayer into the supernatant. With S478A rt-PA this results in the formation of noncovalent complexes that remain associated with the HUVEC monolayer, thereby identifying a large pool of reactive PAI-1 molecules in the monolayer.