Sh Ble Research Articles

Comparison of CRISPR-MAD7 and CRISPR-Cas9 for Gene Disruptions in Komagataella phaffii.

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).

Impact of Different Light Characteristics on the Growth and Lipid Content of Diatom <i>Phaeodactylum tricornutum</i> Transconjugant Strains

Light regulates important metabolic processes in microalgal cells, which can further impact the metabolism and the accumulation of biomolecules such as lipids, carbohydrates, and proteins. Different characteristics of light have been studied on various strains of the model diatom Phaeodactylum tricornutum, but not on transconjugant cells and information on wild-type strains is still limited. Therefore, we studied the impact of different light characteristics such as spectral quality, light intensity and light shift on the growth, and the composition in lipids and fatty acids of P. tricornutum cells to provide a comprehensive context for future applications. Initially, we tested the impact of spectral quality and light intensity on P. tricornutum transformed with an episomal vector (Ptev), harboring the resistance gene Sh ble. Results indicated that Ptev cells accumulated more biomass and overall lipids in spectral quality Red 1 (R1: 34% > 600 nm > 66%) more effectively as compared to Red 2 (R2: 8% > 600 nm > 92%). It was also detected that cell granularity was higher in R1 as compared to R2. Furthermore, by testing two light intensities 65 μmol·m-2·s-1 and 145 μmol·m-2·s-1 light, it was observed that 145 μmol·m-2·s-1 led to an increase in growth trend, total biomass and lipid content. Combining spectral qualities and light intensities, we show that the lipid accumulation raised by 2.8-fold. Studying the light intensity and spectral quality allowed us to optimize the light conditions to R1 spectral quality and light intensity 145 μmol·m-2·s-1. These initial results showed that red light R1 at 145 μmol·m-2·s-1 was the best condition for biomass and total lipids accumulation in Ptev cells. Next, we further combined these two-light optimizations with a third light characteristics, i.e. light shift, where the cultures were shifted during the early stationary phase to a different light environment. We studied Red light shift (Rs) to investigate how light condition variations impacted P. tricornutum transconjugants Ptev and with an episomal vector containing the reporter gene YFP (PtYFP). We observed that Rs induced growth and fatty acid eicosapentaenoic acid (EPA) in Ptev as compared to PtYFP. Altogether, the study shows that red light shift of R1 at 145 μmol·m-2·s-1 promoted biomass and total lipids accumulation in Ptev and PtYFP cells. The study provides a comprehensive approach to using different light characteristics with the aim to optimize growth and lipids, as well as to fatty acid production.

Novel endogenous promoters for genetic engineering of the marine microalga Nannochloropsis gaditana CCMP526

Nannochloropsis is a marine microalga from the Eustigmatophyceae stramenopile lineage that has been studied extensively due to a broad range of industrial applications, mostly related to their oil and pigment production. However, tools to genetically engineer members of this group, and therefore further understand and maximise their industrial potential are still limited. In order to expand the potential industrial uses of this organism, several molecular tools, including gene promoters of different strength, are needed. A comprehensive and diverse set of well-characterized promoters is key to a number of genetic engineering and synthetic biology applications, such as the assembly of complex biological functions or entire metabolic pathways.In this study, we measured the promoter activity of three endogenous constitutive promoters from N. gaditana genes EPPSII (Nga02101); HSP90 (Nga00934); ATPase (Nga06354.1) in driving the expression of a Sh ble- mVenus fluorescent reporter fusion protein. Through a combined approach that includes flow cytometry, RT-qPCR and immunoblotting, we profiled the activity of these promoters at both the transcript and protein level. Two promoters HSP90 (Nga00934) and EPPSII (Nga02101) outperformed the widely used β-tubulin promoter, exhibiting 4.5 and 3.1-fold higher mVenus fluorescence, respectively. A third promoter ATPase (Nga06354.1) was also able to drive the expression of transgenes, albeit at lower levels. We show that the new promoters identified in this study are valuable tools, which can be used for genetic engineering and functional genetics studies in N. gaditana.

Development of endogenous promoters that drive high-level expression of introduced genes in the model diatom Phaeodactylum tricornutum

The marine diatom Phaeodactylum tricornutum is attractive for basic and applied diatom research. We isolated putative endogenous gene promoters derived from genes that are highly expressed in P. tricornutum: the fucoxanthin chlorophyll a/c-binding protein (FCP) C gene, the vacuolar ATP synthase 16-kDa proteolipid subunit (V-ATPase C) gene, the clumping factor A gene and the solute carrier family 34 member 2 gene. Five putative promoter regions were isolated, linked to an antibiotic resistance gene (Sh ble) and transformed into P. tricornutum. Using quantitative RT-PCR, the promoter activities in the transformants were analyzed and compared to that of the diatom endogenous gene promoter, the FCP A gene promoter which has been used for the transformation of P. tricornutum. Among the five isolated potential promoters, the activity of the V-ATPase C gene promoter was approximately 2.73 times higher than that of the FCP A gene promoter. The V-ATPase C gene promoter drove the expression of Sh ble mRNA transcripts under both light and dark conditions at the stationary phase. These results suggest that the V-ATPase C gene promoter is a novel tool for the genetic engineering of P. tricornutum.

Characterizing yeast promoters used in Kluyveromyces marxianus.

Fermentation at higher temperatures can potentially reduce the cooling cost in large-scale fermentation and reduce the contamination risk. Thus, the thermotolerant yeast, Kluyveromyces marxianus, which can grow and ferment at elevated temperatures, is a promising biotechnological tool for future applications. However, the promoters used in K. marxianus are not well characterized, especially at elevated temperatures, which is important in efficient metabolic pathway construction. In this study, six constitutive promoters (P(TDH3), P(PGK), and P(ADH1) from both Saccharomyces cerevisiae and K. marxianus) were evaluated in K. marxianus through the heterologous expression of the KlLAC4, GUSA, and SH BLE genes at various temperatures, with various carbon sources and oxygen conditions. The expression was evaluated at the transcription and protein level using real-time PCR and protein activity determination to eliminate the effect of heterologous protein stability. While the transcription of all the promoters decreased at higher temperatures, the order of their promoting strength at various temperatures with glucose as the carbon source was P(KmPGK) > P(KmTDH3) > P(ScPGK) > P(ScTDH3) > P(KmADH1) > P(ScADH1). When glycerol or xylose was supplied as the carbon source at 42 °C, the order of promoter strength was P(KmPGK) > P(ScPGK) > P(KmADH1) > P(ScADH1) > P(ScTDH3) > P(KmTDH3). The promoter activity of P TDH3 decreased significantly, while the promoter activity of both of the P(ADH1) promoters increased. Oxygen conditions had non-significant effect. The results of this study provide important information for fine-tuned pathway construction for the metabolic engineering of K. marxianus.

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii.

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.

Microbial Inactivation of Pseudomonas putida and Pichia pastoris Using Gene Silencing

Antisense deoxyoligonucleotide (ASO) gene silencing was investigated as a potential disinfection tool for industrial and drinking water treatment application. ASOs bind with their reverse complementary mRNA transcripts thereby blocking protein translation. While ASO silencing has mainly been studied in medicine, it may be useful for modulating gene expression and inactivating microorganisms in environmental applications. In this proof of concept work, gene targets were sh ble (zeocin resistance) and todE (catechol-2,3-dioxygenase) in Pichia pastoris and npt (kanamycin resistance) in Pseudomonas putida. A maximum 0.5-fold decrease in P. pastoris cell numbers was obtained following a 120 min incubation with single-stranded DNA (ssDNA) concentrations ranging from 0.2 to 200 nM as compared to the no ssDNA control. In P. putida, a maximum 5.2-fold decrease was obtained after 90 min with 400 nM ssDNA. While the silencing efficiencies varied for the 25 targets tested, these results suggest that protein activity as well as microbial growth can be altered using ASO gene silencing-based tools. If successful, this technology has the potential to eliminate some of the environmental and health issues associated with the use of strong chemical biocides. However, prior to its dissemination, more research is needed to increase silencing efficiency and develop effective delivery methods.

Decrease of hirudin degradation by deleting the KEX1 gene in recombinant Pichia pastoris

Our previous study on recombinant hirudin production in Pichia pastoris demonstrated that, although the total productivity of hirudin was fairly high, its degradation was still severe, even if many engineering methods were applied to improve cell viability and reduce the release of intracellular proteinases. In this work, a pop-in/pop-out method, replacing the auxotrophic marker ARG4 gene with the resistant marker sh ble gene, was used to delete the KEX1 gene to reduce hirudin degradation in P. pastoris GS115Hir. Using this strategy, hirudin degradation was greatly decreased. At the same wet cell weight and cell viability, the percentage of intact hirudin Hir65 in total hirudin in strain GS115HirDeltakex1 was always kept as high as 90% in the initial stage of the methanol fermentation phase and above 62% even in the later stage of the methanol fermentation phase, whereas the percentage for the undeleted strain GS115Hir was only about 40% in the whole methanol fermentation phase. As a result, the intact hirudin Hir65 concentration could maximally reach 2.4 g/l in GS115HirDeltakex1 while it was only 1.1 g/l in GS115Hir.

Cloning of a Cholesterol-α-glucosyltransferase from Helicobacter pylori

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase.

Sh ble and Cre adapted for functional genomics and metabolic engineering of Pichia stipitis

Pichia stipitis is widely studied for its capacity to ferment d-xylose to ethanol. Strain improvement has been facilitated by recent completion of the P. stipitis genome. P. stipitis uses CUG to code for serine rather than leucine, as is the case for the universal genetic code thereby limiting the availability of heterologous drug resistance markers for transformation. Development of a modified selectable marker for resistance to bleomycin ( Sh ble) and efficient excision of the marker after integration ( loxP/Cre) should facilitate functional genomics and metabolic engineering in this yeast. The Sh ble marker did not code for an active protein in P. stipitis until four CUG codons were mutagenized to TTG, which is properly translated as leucine in yeasts that use the alternative yeast nuclear genetic code. The 18 CTG codons in Cre were mutagenized in a similar manner and the system was used to delete XYL2. The resulting xyl2Δ mutant did not use xylose as a carbon source.

Sequential gene deletions in Hypocrea jecorina using a single blaster cassette

In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5'-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4-1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10-13% of the transformants in the deletion of one of the two kinase genes. For excision of the pyr4 blaster cassettes, Deltaglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent transformation of one of these auxotrophic Deltaglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Deltaglk1 strains showed reduced growth on d-glucose and d-fructose whereas Deltahxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Deltaglk1Deltahxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose.

Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells

Zeocin is a member of the bleomycin/phleomycin family of antibiotics, known to bind and cleave DNA. We established human SK-OV-3 cells that stably express the Zeocin resistance gene (Sh ble) using an ecdysone-inducible mammalian expression system. Surprisingly, our results demonstrated that Zeocin, added in the culture medium to maintain the expression of the ecdysone receptor, was responsible for the formation of DNA strand breaks in the recombinant cells. This suggests that the Zeocin is not completely detoxified and is still able to cleave DNA, despite the stable expression of the Sh ble gene in the recombinant clones. Our study indicates that one needs to be very cautious in the interpretation of data involving stable cell lines selected with Zeocin.

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha‐T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcpA promoter. pPha‐T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat‐1 genes confer resistance to the antibiotic nourseothricin, and nptII confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uidA and gfp were also introduced into P. tricornutum using pPha‐T1. Gus expression in some transformants reached 15 μg·μg−1 of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum, enhancing the potential of this organism for exploring basic biological questions and industrial applications.

Transformation of Nonselectable Reporter Genes in Marine Diatoms

: We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations. Transformation efficiencies were around 10(-6), and individual cell lines could be maintained at -80 degrees C following cryopreservation. Also, P. tricornutum could be transformed simultaneously with two different plasmids, one containing the Sh ble gene and another containing the firefly luciferase gene (LUC) under control of a promoter derived from a fucoxanthin, chlorophyll a/c-binding protein gene (FCP). In these cotransformants, LUC activity was light inducible. The transient transformation of the centric diatom Thalassiosira weissflogii with the bacterial beta-glucuronidase (GUS) gene has also been achieved using similar transformation technology. The availability of gene transfer protocols for marine diatoms, together with a range of functional reporter genes and regulated expression systems, will permit molecular dissection of their biology and allow an assessment of the biotechnological potential of these organisms.

Expression of heterologous genes in Schizophyllum commune is often hampered by the formation of truncated transcripts.

GPD regulatory sequences were used to express a phleomycin resistance gene (Sh ble) in Schizophyllum commune, resulting in high numbers of phleomycin-resistant transformants. Attempts to express heterologous genes coding for hygromycin B phosphotransferase (hph), aminoglycoside phosphotransferase (apt), beta-glucuronidase (uidA) and beta-galactosidase (lacZ) using the same regulatory sequences were not successful and no mRNA could be detected. Cloning the hph and uidA genes in an internally deleted GPD gene resulted in truncated transcripts which ended within the 5'-parts of the heterologous genes. Cloning of the same genes as transcriptional fusions downstream from the Sh ble gene also resulted in truncated transcripts ending in the 5'-parts of these heterologous genes. It is suggested that AT-rich sequences in heterologous genes might be involved in generating these truncated transcripts, thereby preventing expression in S. commune.

Fusion of green fluorescent protein with the Zeocin-resistance marker allows visual screening and drug selection of transfected eukaryotic cells.

Green fluorescent protein (GFP) and the Zeocin-resistance gene Sh ble (ZeoR) were fused together to generate a bifunctional protein for the identification and selection of transfected mammalian cells. Expression of this hybrid selectable marker, GFP-ZeoR, was visually detected and conferred Zeocin resistance in prokaryotes and eukaryotes. This selectable marker provides a way to determine transient transfection efficiencies in tissue culture cells using fluorescence microscopy. Expression of the GFP-ZeoR was also used to identify and select stable mammalian cell lines expressing a heterologous gene. Selection was efficient and GFP fluorescence provides an excellent, noninvasive technique to monitor the success of Zeocin selection during the development of the stable cell lines. This hybrid resistance gene combines the functional properties of the Zeocin-resistance marker and GFP and should be useful for combined selection and fluorescence in a variety of organisms.

Unexpected behavior of a gene trap vector comprising a fusion between the Sh ble and the lacZ genes

A new gene trap vector has been designed, comprised of a fusion between the Sh ble gene, which confers resistance to the antibiotic phleomycin, and the lacZ gene (phleal fusion gene). A synthetic splice acceptor, made of the yeast branchpoint followed by a pyrimidin-rich sequence of 27 nucleotides, is included at the 5′ extremity. The linearized gene trap vector was introduced into mouse embryonic stem cells (ES cells), and 40 phleomycin resistant (phleAL) cell lines possessing a single copy of the insert were selected. They were stable in expressing the lacZ gene. Reporter gene expression was studied at days 8.5 and 10.5 of embryonic development in chimeric embryos obtained after injection of phleoL ES clones into 8-cell stage embryos. Out of 20 phleal lines examined, 14 exhibited β-galactosidase expression at day 10.5. Use of the phleal fusion gene trap vector to select genes expressed in ES cells, therefore, is compatible with the isolation of genes expressed at midgestation. However, and most intriguingly, 10 out of these 14 cell lines (71%) displayed reporter gene expression mostly in heart and liver. Two of them exhibited, in addition, expression in central nervous system (CNS) or in CNS and limb buds, respectively. Germline chimeras were subsequently obtained and 15 mouse lines have been established. Intercrosses of animals heterozygous for the insertion revealed a mutant phenotype in several lines. © 1996 Wiley-Liss, Inc.

Stable nuclear transformation of the diatom Phaeodactylum tricornutum.

A nuclear transformation system has been developed for the diatom Phaeodactylum tricornutum using microparticle bombardment to introduce the sh ble gene from Streptoalloteichus hindustanus into cells. The sh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from the P. tricornutum fcp genes were used to drive expression of sh ble. Between 10-100 transformants were recovered/10(8) cells. Transformants were able to grow on at least 500 micrograms/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations the sh ble gene was present in 1-3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1-2.0 micrograms/mg total protein). The cat reporter gene fused to a fcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed in P. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.

Generation of Small Fusion Genes Carrying Phleomycin Resistance andDrosophilaAlcohol Dehydrogenase Reporter Properties: Their Application in Retroviral Vectors

We have used theDrosophilaADH cDNA to engineer new fusion genes carrying both reporter activity and bleomycin/phleomycin resistance (Sh ble). Cassettes of ADH::Sh ble, Sh ble::ADH, or ADH::Sh ble::ADH with or without polyadenylation signals were constructed. Placed under the control of the strong CMV promoter, these constructs induced intense ADH substrate staining and phleomycin resistance, whatever the position of the ADH gene, in avian or mammalian cell lines. SNV-based nonreplicative retroviral vectors were constructed and introduced into the appropriate packaging cell line. Titers up to 106ADH forming units/ml of viral supernatant were obtained except for the ADH::Sh ble::ADH construct, which reached 105ADH forming units. These retroviral vectors were inoculated to the E3 chick embryo via the coelom. Three days later, cells from different organs were put in culture for 24 h and stained to detect ADH activity. A large number of positive cells were found in cultures from all organs. The new fusion genes described here are, to our knowledge, the smallest (1.1 kb) published to date that carry both reporter and drug resistance properties. These genes represent the basis of a new retroviral vector model with three distinct properties in two genetic units; their advantage is to reduce the size and increase the efficiency of the vector.

The ble resistance gene as a new selectable marker for Trypanosoma brucei: fly transmission of stable procyclic transformants to produce antibiotic resistant bloodstream forms.

We describe here the stable transformation of Trypanosoma brucei using a new selectable marker for kinetoplastid protozoa, the Sh ble, or phleomycin, resistance gene. A plasmid containing this gene targeted to the tubulin gene locus by homologous sequences was introduced into procyclic trypanosomes by electroporation and cells selected for antibiotic resistance. Southern analysis of stable transformants showed that the plasmid had been integrated into the tubulin locus by homologous recombination. Analysis of bloodstream stage transformants, produced by transmission through the vector Glossina, showed that the resistance gene was conserved and expressed in these forms in the absence of selective drug pressure. In both procyclic and bloodstream forms, transcription of the ble gene appears to originate from the upstream tubulin promoter, despite the presence of a VSG promoter in the integrated construct. The generation of stable bloodstream transformants for the first time will facilitate the study of gene function and expression during the trypanosome life cycle, and aid in the investigation of genetic exchange in these organisms.