A phosphoglycerate kinase-related gene conserved between Trypanosoma brucei and Crithidia fasciculata.

Trypanosoma brucei and Crithidia fasciculata both contain three different phosphoglycerate kinase (PGK) genes, A, B and C, in a tandem array. The genes B and C encode the major PGKs: the cytosolic and glycosomal PGKs, respectively. The PGK-A genes of both Trypanosomatid species encode open reading frames related to PGK, which have most active site residues conserved, but contain an insert of 80 amino acids at approximately position 80 of the 420 amino acids average PGK sequence. The deduced amino acid sequence of these inserts is conserved between T. brucei and C. fasciculata (48% positional identity), indicating its functional importance. Although we have not been able to demonstrate PGK activity in the PGK-A gene product, we consider it likely that this gene codes for a minor PGK with special function.     

Primary structure of Trypanosoma cruzi small-subunit ribosomal RNA coding region: comparison with other trypanosomatids

A Trypanosoma cruzi small subunit ribosomal RNA gene was sequenced from genomic recombinant plasmid clones. The assigned coding region was 2319 bp, the longest SSU rRNA gene described to date. On the basis of comparisons with published sequences from Crithidia fasciculata, Trypanosoma brucei, and Leishmania donovani, we conclude that the extra nucleotides in the T. cruzi gene occur in highly variable regions of rRNA genes. A phylogenetic analysis was performed with the SSU rRNA sequences from these four trypanosomatids and from Euglena gracilis as an outgroup. The Leishmania and Crithidia sequences were remarkably similar to each other (not separable statistically). Given the standard errors associated with the branching order of the two Trypanosoma species and the Leishmania-Crithidia branch, the actual topology cannot be unequivocally determined using only the ribosomal sequences analyzed here.     

Production and characterization of human 293 cell lines expressing the site-specific recombinase Cre

We have constructed 293 cell lines expressing the site-specific Cre recombinase from bacteriophage P1, that acts on a 34 bp target sequence calledloxP. Stably transformed cells were obtained by transfection with a plasmid containing Cre and a selectable marker under the control of viral promoters. The resulting 293 Cre cell lines could be used to induce expression from adenovirus vectors containing reporter genes under the control of a Cre responsive “molecular switch” High efficiency recombination was observed for Ad viral DNA containingloxP sites. The Cre expressing cell lines described here are likely to be useful for several purposes: For expression of toxic gene products from Cre inducible viral vectors, to induce recombination betweenloxP sites in transfected plasmids, and to induce deletions or rearrangements of genes defined byloxP sites in viral genomes.     

Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum

The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.     

Stable DNA transfection of a wide range of trypanosomatids

We have shown that the Leishmania major transfection vector pR-NEO (or derivatives thereof) can be introduced and stably maintained in four species complexes of pathogenic Leishmania (L. tropica, L. mexicana, L. donovani, L. braziliensis), and the genera Endotrypanum and Crithidia; transfection of Trypanosoma cruzi or Trypanosoma brucei was not successful. Quantitative plating assays showed that the transfection efficiencies were high in L. major and Leishmania amazonensis (5x10(-5)/cell) and about 10-fold less for Leishmania panamaensis and Crithidia. Leishmania donovani transfected with pR-NEO retained the ability to infect hamsters, and amastigotes recovered after 2 months yielded G418-resistant promastigotes which retained high levels of extrachromosomal pR-NEO DNA. In promastigotes, the transfected DNA existed as extrachromosomal circles, and expressed the predicted 2.4-kb hybrid NEO/DHFR-TS mRNA bearing the trans-spliced miniexon. Large quantitative differences were observed only in Crithidia: relative to transfected Leishmania species, the copy number of pR-NEO was elevated 20-fold, while the levels of the NEO/DHRFR-TS mRNA or Escherichia coli beta-galactosidase (synthesized from the expression vector pX-beta GAL) were reduced 80 and more than 1000-fold, respectively. Thus, genetic signals derived from L. major DNA that mediate RNA expression or stability are recognized by the heterologous Leishmania species but less efficiently by Crithidia. These studies suggest that pR-NEO derived vectors may be applied to the study of genes expressed throughout the life cycle in a wide range of pathogenic trypanosomatids.     

3' UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum

In Plasmodium parasites the fusion of gametes to form a fertilized zygote and morphogenesis into the motile ookinete are critical developmental stages in the parasite's complex life cycle. In analogous developmental stages of metazoan organisms 3' gene flanking regions are critical in the regulation of gene expression. To determine whether these mechanisms are conserved in the protozoan parasite we studied the 3' gene flanking elements necessary for the expression of Pgs28, the major surface protein of mature zygotes and ookinetes of the chicken malaria Plasmodium gallinaceum. The DNA sequence of the pgs28 3' gene flanking region contains 7 eukaryotic polyadenylation consensus signals (AATAAA/ATTAAA). An unusual 82% T-rich region is located 55 nucleotides upstream of the fifth polyadenylation signal (ATTAAA). The pgs28 mRNA terminates approximately 20 nucleotides from the polyadenylation signal in a poly (A) tail. To determine whether the T-rich region and polyadenylation signals were necessary for Pgs28 protein expression, sexual stage parasites were transfected with plasmids containing deletions of these elements utilizing firefly luciferase (LUC) and beta-glucuronidase (GUS) as markers of transient gene transfection. The parasites were allowed to develop in vitro to the ookinete stage and assayed for enzymatic activity. Cells transfected with plasmids containing deletions of the T-rich region or fifth eukaryotic polyadenylation consensus signal expressed 89 and 92%, less enzymatic activity respectively than those transfected with the full length pgs28 3' gene flanking region. The U-rich element and fifth eukaryotic polyadenylation consensus sequence within the pgs28 3' UTR are therefore necessary for Pgs28 protein expression.     

Expression and rescue of a nonselected marker from an integrated AAV vector

We used rep+ and rep- recombinant AAV-plasmid vectors containing the nonselectable marker chloramphenicol acetyltransferase (CAT) driven by the AAV p40 promoter, and having a selectable marker, neo, inserted in the plasmid genome, and driven by a herpesvirus thymidine kinase gene promoter. Each vector was transfected into human 293 cells or HeLa cells and the neo gene was used to select geneticin-resistant (genr) cells containing integrated vectors. The genr cells were then screened for expression of the unselected marker CAT. For 293 cells, most clones from the rep- vector gave high CAT expression whereas only 50% of those from the rep+ vector expressed CAT, generally at low level. For HeLa cells about 25% of the clones derived from either the rep+ or rep- vector expressed CAT, and several clones from the rep+ vector gave very high yields. We also analyzed integrated rep+ vectors by rescue after superinfection with adenovirus and by Southern blotting. The AAV-CAT genome could be rescued from 50% of HeLa cell clones but not from 293 cell clones. Lack of rescuability reflected rearrangement of the AAV genome termini or the rep gene. Western blotting showed low level constitutive expression of rep protein in one 293 cell clone and two HeLa cell clones. Thus, the AAV p40 promoter (as well as p5 and p19) can function in integrated vectors to express unselected markers which can subsequently be rescued. Expression and rescue depended upon several parameters including the cell type, the initial structure of the vector (rep+ or rep-) but not continued expression of rep, and possibly global effects of the surrounding chromatin.     

Transient marker stabilisation: a general procedure to construct marker-free recombinant vaccinia virus

Summary.  Recombinant vaccinia viruses based on the highly attenuated Modified Vaccinia Ankara (MVA) strain expressing HIV-1 antigen genes were constructed by a novel procedure involving the transient use of two marker genes. The selectable markers used, the Escherichia coli guanine phosphoribosyltransferase (gpt) and the ?-galactosidase (lacZ) genes, are not retained within the final recombinant virus. The transient marker stabilisation (TMS) procedure allows the generation of marker-free recombinant viruses in a series of simple plaque purification steps. HIV-l gag pol genes were inserted into two loci of vaccinia MVA demonstrating the efficiency of the procedure. Accepted November 10, 1997 Received September 14, 1997     

High level expression of hepatitis B virus surface antigen in stably transfected Drosophila Schneider-2 cells.

Two transfer vector systems have been constructed for the generation of Drosophila melanogaster Schneider-2 (DS-2) cells transfected stably and used to express the small surface antigen of hepatitis B virus (HBsAg). One system is based on the cotransfection of an expression vector for the S gene under the control of an inducible Drosophila metallothionein (Mtn) promotor and a resistance plasmid which carries a selectable marker dihydrofolate reductase (dhfr) gene under the control of a Drosophila actin 5C distal promoter. The second system is based on the transfection of a single plasmid, which includes both expression units. Both vector systems were suitable for the generation of stably transfected DS-2 cell-lines secreting high levels of HBsAg. The quantities of HBsAg expression from polyclonal DS-2 cells correlated strictly with the concentration of the transfected S gene expression vector. Clonal cell-lines selected from the most efficient HBsAg producing polyclonal cell-populations were examined in more detail. All of the transfected S genes were found to be integrated and the copy numbers per genome varied extremely between 10 and 240. Furthermore, the levels of secreted HBsAg varied greatly between different clones and in best they reached up to 7 microg/ml under serum-free cell culture conditions. Thus, DS-2 cells transfected stably provide an alternative source for the production of HBsAg particles for diagnostic purposes and vaccine development.     

Identification of an intergenic region that is not essential for replication of goatpox virus

A recombinant goatpox virus was constructed in which an enhanced green fluorescent protein gene was inserted under the control of the 11K late promoter, a guanine phosphoribosyltransferase gene was inserted under the control of the 7.5K early/late promoter, and exogenous genes were inserted into an intergenic region between loci gp_24 and gp_24.5 of the recombinant genome. Analysis of protein expression showed that LT cells infected with only the recombinant virus produced specific fluorescence. A comparative growth assay demonstrated the stability of the recombinant virus at the replication level. These results suggest that the intergenic region is not essential for replication of goatpox virus.     

不同类型的HBsAg真核表达质粒在EBV永生化B淋巴母细胞中的表达

目的 探讨3种不同类型的HBsAg真核表达质粒在EBV永生化B淋巴母细胞中的表达。方法 用3种不同类型的质粒载体分别构建乙型肝炎表面抗原（HBsAg)真核表达质粒（pCI-S,pMEP4-S,pLXSN-S)；然后用阳离子脂质体介导的转染法分别转染正常人EBV永生化的B淋巴母细胞，经G418或HygromycinB筛选出抗性细胞克隆，用RT-PCR检测抗性细胞总RNA中目的基因在转录水平的表达；用ELISA检测抗性细胞培养上清和细胞裂解液中HB-sAg的含量。结果 3种不同类型HBsAg重组表达质粒转染的EBV永生化B淋巴母细胞，在转录水平上，可检出HB-sAgmRNA的表达；在蛋白水平上，细胞增养上清和细胞裂解液均可检出HBsAg；而以EB病毒表达质粒pMEP4-S表达最高，真核表达质粒pCI-S和逆转录病毒表达质粒pLXSN-S表达HBsAg的量无明显差异。结论 3种不同类型的HBsAg真核表达质粒均可在EBV永生化B淋巴母细胞中稳定表达，EB病毒表达质粒pMEP4-S表达的HBsAg明显高于真核表达质粒pCI-S和逆转录病毒表达质粒pLXSN-S。     

A transformation vector for stage-specific expression of heterologous genes in Trypanosoma cruzi epimastigotes

To express heterologous genes in a stage-specific manner, we constructed a transformation vector for Trypanosoma cruzi containing a selection gene (hyg) and a reporter gene (luc) flanked by sequences of the multicopy 1f8 gene arranged so as to provide a trans-splicing acceptor site to hyg and a putative polyadenylation signal to luc. The intergenic region of the T. cruzi genes 294 and KAP was placed between hyg and luc, contributing the polyadenylation signal of 294 to hyg and the KAP trans-splicing acceptor site to luc. Transformation was carried out by electroporation, and transformed epimastigotes were selected in medium containing hygromycin B. Through double homologous recombination of the 1f8 sequences with their chromosomal counterparts, the construction is inserted into the 1f8 locus, substituting probably one and no more than a few copies of the 1f8 gene without having apparent deleterious effects on the parasite. cDNA analysis demonstrated that the introduced signals were correctly processed, resulting in translatable hyg and luc mRNAs. Whereas epimastigotes express luciferase, no expression is found in the trypomastigote stage.     

Green fluorescent protein as a selectable marker of retrovirally transduced hematopoietic progenitors.

Recombinant retroviruses are most commonly used in hematopoietic stem cell gene therapy trials, but gene transfer efficiency is still inadequate with the present vectors. One approach for overcoming this problem is to develop methods of selecting and enriching the successfully transduced cells. We investigated the feasibility of using the green fluorescent protein (GFP) gene as a selectable marker of hematopoietic cells. When M1 murine leukemia cells were electroporated with GFP expression vectors, a red-shifted mutant (S65T) GFP showed several-fold greater fluorescence than the wild-type GFP and generated readily detectable green light under control of SRalpha or CAG promoter. We then inserted an SRalpha-S65T GFP cassette into the MSCV retrovirus vector and established virus producer cells. Infection of primary murine bone marrow cells resulted in a distinct population with green fluorescence, which was separated by fluorescence-activated cell sorting. The fractionated bright cells gave rise to fluorescent spleen colonies in lethally irradiated mice, while the fluorescence-negative cells yielded only dark colonies. These results indicated that GFP is a faithful marker in gene transfer into hematopoietic progenitor/stem cells, facilitating selection of the transduced cells and tracking of their progeny in vivo.     

9.1C3胞内抗体基因的克隆及其对9.1C3 分子表达的抑制作用

目的 获得9．1C3胞内抗体基因的真核表达载体,观察胞内抗体对9．1C3分子表达的抑制作用,为进一步研究9．1C3分子对NK细胞功能的影响打下基础。方法 设计引物,以9．1C3 ScFv基因为模板进行PCR扩增,引入蛋白质内质网定位所需的信号肽、KDEL以及作为表达检测标记的Etag序列。回收纯化PCR产物,酶切后连接到pUC19载体上,并进行序列测定。序列正确后切下胞内抗体基因片段,重组到真核表达载体pRc/CMV中,酶切鉴定后用DEAE－dextran方法瞬时转染真核细胞COS7,用胞内染色和Western blot方法检测胞内抗体的表达。接着用Lipofectamine把胞内抗体基因转入9．1C3阳性的Jurkat细胞,用FCM观察胞内抗体对9．1C3分子表达的影响。结果 DNA序列测定证明,通过PCR成功地为9．1C3 ScFv引入了内质网定位所需的信号肽、KDEL及E－tag序列,成功地构建了胞内抗体真核表达载体,通过胞内染色方法和Western blot证明胞内抗体在COS7中得到表达。转入Jurkat细胞后发现胞内抗体能下调9．1C3分子的表达水平。结论 以9．1C3 ScFv基因为模板PCR扩增得到9．1C3胞内抗体基因,并构建了真核表达载体。     

Rescue of Targeted Regions of Mammalian Chromosomes by in Vivo Recombination in Yeast

In contrast to other animal cell lines, the chicken pre-B cell lymphoma line, DT40, exhibits a high level of homologous recombination, which can be exploited to generate site-specific alterations in defined target genes or regions. In addition, the ability to generate human/chicken monochromosomal hybrids in the DT40 cell line opens a way for specific targeting of human genes. Here we describe a new strategy for direct isolation of a human chromosomal region that is based on targeting of the chromosome with a vector containing a yeast selectable marker, centromere, and an ARS element. This procedure allows rescue of the targeted region by transfection of total genomic DNA into yeast spheroplasts. Selection for the yeast marker results in isolation of chromosome sequences in the form of large circular yeast artificial chromosomes (YACs) up to 170 kb in size containing the targeted region. These YACs are generated by homologous recombination in yeast between common repeated sequences in the targeted chromosomal fragment. Alternatively, the targeted region can be rescued as a linear YACs when a YAC fragmentation vector is included in the yeast transformation mixture. Because the entire isolation procedure of the chromosomal region, once a target insertion is obtained, can be accomplished in ~1 week, the new method greatly expands the utility of the homologous recombinationproficient DT40 chicken cell system.