Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.     

The hygromycin-resistance-encoding gene as a selection marker for vaccinia virus recombinants.

Hygromycin B (Hy), an inhibitor of RNA translation, was shown to block the replication of vaccinia virus (VV) in cultured cell lines. Insertion of the Escherichia coli Hy resistance-encoding gene (hph) into the VV genome under control of early or late synthetic VV promoters could overcome inhibition of viral replication. When hph was inserted into VV in tandem with the human papillomavirus type 16 (HPV16) L1 open reading frame, hph recombinant viruses could be selected which expressed HPV16 L1.     

Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase

The susceptibilities of the protozoan parasites Leishmania mexicana and Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 microg ml(-1) was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T. brucei in vitro. The gene encoding blasticidin S deaminase (BSD) was therefore incorporated into cassettes for targeting to the cysteine proteinase C locus of L. mexicana (CPC::BSD) and the tubulin locus of T. brucei (tub::RAD51-BSR). Following transfection of mutant parasites that contained other well-established selectable marker genes (HYG, NEO, BLE, PAC and SAT), clones resistant to 10 microg ml(-1) blasticidin S were shown by PCR and Southern blotting to have integrated the cassettes by homologous recombination. The results confirm that BSD can be used as a selectable marker gene for targeted chromosomal integration during genetic manipulations of trypanosomatids.     

Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

胸苷激酶基因治疗胃癌的体外实验

将单纯疱疹病毒胸苷激酶基因（ＨＳＶ－ｔｋ）导入恶性肿瘤细胞,随后可应用药物丙氧鸟苷（ｇａｎｃｉｃｌｏｖｉｒ, ＧＣＶ）选择性杀死肿瘤细胞．构建了含胸苷激酶与潮霉素磷酸转移酶（ｈｐｈ）融和基因（ＨｙｔＫ）的真核表达载体ＬＸｐｓｐ－ＨｙｔＫ．以脂质体（ｌｉｐｏｆｅｃｔｉｎ）为介导,将这种质粒与仅含潮霉素Ｂ基因的质粒ＬＸＳＨ 分别转染胃癌细胞系ＢＧＣ－８２３,用６０ Ｕ／ｍ ｌ潮霉素Ｂ进行筛选,得到了可稳定传代的阳性克隆,分别命名为ＢＧＣ－ＨｙｔＫ 和ＢＧＣ－Ｈｙ．三种细胞的生长曲线无明显差别．用不同浓度的ＧＣＶ 分别作用于ＢＧＣ－ＨｙｔＫ, ＢＧＣ－Ｈｙ 及ＢＧＣ－８２３,０．０２～２００ μｇ／ｍ ｌ 的ＧＣＶ 对ＢＧＣ－ＨｙｔＫ 细胞有明显的杀伤作用（ＩＣ５０＝ ０．０２ μｇ／ｍ ｌ）,而对另外两种细胞几乎无毒性作用（ＩＣ５０＞ ２００μｇ／ｍ ｌ）．２０ μｇ／ｍ ｌＧＣＶ 作用９６ ｈ 后,仅存在２０％ 的ＢＧＣ－ＨｙｔＫ 就可使周围的大部分ＨＳＶ－ｔｋ－ 的肿瘤细胞死亡,说明存在较显著的“旁观者效应”     

Site-independently integrated transgenes in the elite restorer rice line Minghui 63 allow removal of a selectable marker from the gene of interest by self-segregation

In this study, we have demonstrated that two independent loci are involved in the integration of the insecticidal protein gene cryIAb/cryIAc and selectable marker gene hph in the recipient genome of the elite Chinese CMS restorer line Minghui 63. We have also documented the structural organization of these transgenes in each locus by restriction enzyme digestion and Southern blot analysis. The independent locus integration of different transgenes allowed us to remove the selectable marker gene hph from the gene of interest simply by self-segregation. Not having the selectable marker gene will enhance the commercial value of our transgenic line TT51-1, which showed a consistently high level of resistance against repeated infestations of yellow stem borers and natural outbreaks of leaf-folders, without a reduction in yield potential.     

Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T(0) plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T(1) generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T(2) plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T(2) plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.     

PEG介导的猴头菌遗传转化体系的建立

对猴头菌Hericium erinaceus原生质体制备的各种因素进行比较研究,结果表明,猴头菌原生质体制备的最佳体系为：液体培养5d的猴头菌丝,以0.6mol/L KCl作为稳渗剂,加入含1.0%纤维素酶+1.0%蜗牛酶+1.0%溶壁酶的复合酶,在30℃酶解猴头菌丝3h时,原生质体得率达到3.0×106个/mL。潮霉素敏感性测试表明,猴头菌在PDSA固体培养基上的潮霉素最低筛选浓度为60μg/mL。采用PEG介导的原生质体法,将质粒pBgGI-hph（含有灵芝gpd1-Gl启动子和潮霉素抗性基因hph）转化猴头菌原生质体,经潮霉素初步筛选以及PCR鉴定,表明有4株猴头菌拟转化子的基因组扩增出hph基因;转化子经过多次转接后进行Southern杂交验证,结果表明4个转化子的基因组中均稳定整合了hph抗性基因。     

Rapid transformation of high cellulase-producing mutant strains of Trichoderma reesei by microprojectile bombardment

Intact conidia of three industrially relevant strains of Trichoderma reesei were effectively transformed by particle bombardment. Transformations were carried out individually with plasmids carrying either the fungal amdS or bacterial hph gene as a selectable marker and by cotransformation with both plasmids. Transformant yields with single plasmids were up to 11 stable transformants per microg DNA at the bombardment distance of 6 cm. Mitotic stability of the transformants was 75-100% and the cotransformation efficiency averaged 92% when the first selection was performed on hygromycin B plates. The entire procedure could be completed in 1 week with the hph marker.     

黑曲霉（Aspergillus niger）启动子的克隆及其序列特征

应用启动子筛选载体（Ｐｒｏｍｏｔｅｒ－ｔｒａｐｖｅｃｔｏｒ）ＰＬＸ２Ａ构建了１个黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓｎｉｇｅｒ）基因组文库,这个载体含有１个潮霉素Ｂ（Ｈｙ）磷酸转移酶编码基因（ｈｐｈ）它能以启动子活性选择ＤＮＡ片段,用这个基因组文库转化大肠杆菌,获得了８００００转化子菌落,其中９４％含有插入片段,用这个基因库的质粒ＤＮＡ转化黑曲霉,产生了５３个抗潮霉素Ｂ（Ｈｙ＾Ｒ）菌落,２１个转化子的Ｓ     

Ribosomal protein L25 from Trypanosoma brucei: phylogeny and molecular co-evolution of an rRNA-binding protein and its rRNA binding site.

The gene encoding ribosomal protein L25, a primary rRNA-binding protein, was isolated from the protozoan parasite Trypanosoma brucei. Hybridization studies indicate that multiple copies of the gene are present per T. brucei haploid genome. The C-terminal domain of L25 protein from T. brucei is strikingly similar to L23a protein from rat, L25 proteins from fungal species, and L23 proteins from eubacteria, archaebacteria, and chloroplasts. A phylogenetic analysis of L23/25 proteins and the putative binding sites on their respective LSU-rRNAs (large subunit rRNAs) provides a rare opportunity to study molecular co-evolution between an RNA molecule and the protein that binds to it.     

Reduction in solanapyrone phytotoxin production by Ascochyta rabiei transformed with Agrobacterium tumefaciens

Agrobacterium tumefaciens was used to transform Ascochyta rabiei, the causal agent of chickpea blight. Employing a T-DNA containing a hygromycin resistance gene (hph), 908 transformants were obtained from germinated pycnidiospores on a selective medium containing hygromycin. Transformants were confirmed using PCR and Southern analyses and of four of these that were tested, two had integrated multicopies of the hph gene, one had two copies and one had a single insertion. Transformants were tested for the production of solanapyrone A toxin using a microtitre plate assay. Loss of toxin production by transformants was confirmed by reversed phase high-performance liquid chromatography. Sixteen transformants out of 668 tested produced significantly less solanapyrone A than the wild-type strain.     

Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha

The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.