Overexpression of chitinase gene with a GC-rich synthetic enhancer in tobacco plant (Nicotiana tabacum L.) Overekspresi gen kitinase dengan enhancer sintetis kaya GC pada tanaman tembakau (Nicotiana tabacum L.)

Ringkasan Perakitan tanaman perkebunan toleran terhadap serangan cendawan patogenik dilakukan dengan mengoverekspresikan gen penyandi kitinase. Untuk itu elemen DNA peningkat ekspresi (enhancer E52) yang berupa oligonukleotida 52 pb dan kaya kandungan basa purin (GC) disisipkan di ujung 5’ konstruk 35S-chi. Penyisipan E52 tersebut dilakukan secara lebih terarah pada situs ganda HindIII-SalI dari MCS pCAMBIA2301. Melalui situs HindIII yang terletak tepat di ujung 3’ E52, konstruk 35S-chi kemudian disambungkan dengan E52 pada pCAMBIA tersebut. Transformasi DNA rekombinan ke dalam sel tembakau dikerjakan melalui perantaraan Agrobacterium tumefaciens LBA4404. Sel tanaman transgenik diseleksi dan diregenrasi dalam media yang mengandung 3% sukrosa, 0,5 mg/L diregenerasikan pada media MS padat benzilaminopurin (BAP) dan 50 mg/L kanamisin. Pada media ini tunas transgenik tembakau mulai terbentuk setelah 5 minggu penanaman. Analisis tingkat aktivitas enzimatis menunjukkan bahwa aktivitas kitinase pada tembakau transgenik 40 hingga 80 kali lebih tinggi daripada non-transgenik. Pengujian hibridisasi protein menggunakan antibodi anti-kitinase, dot blot dan western blot, membuktikan bahwa enhancer tersebut dapat meningkatkan ekspresi transgen chi pada tanaman tembakau.Summary Development of estate crops tolerant to pathogenic fungi is conducted by overexpressing chi gene. For this purpose, a synthetic enhancer consisting of 52 base pairs and GC-rich was inserted at immediate 5’ end of a 35S-chi cassette. Insertion of the E52 was directed at HindIII-SalI restriction sites of the pCAMBIA2301 MCS. With HindIII restriction site located just after the 3’ end of the E52 sequence, the 35S-chi construct was then ligated with the E52 of the pCAMBIA. Transformation of the resulting recombinant DNA into tobacco cells was mediated by Agrobacterium tumefaciens LBA4404. The transgenic cells were selected and regenerated on a solid MS medium supplemented with 3% sucrose, 0.5 mg/L benzylamino purine (BAP) and 50 mg/L kanamycin. Tobacco shoots were initiated after 5 weeks inoculation on the selection media. Enzymatic analysis demonstrated that chitinase activity of transgenic tobacco was 40 to 80 folds higher than that of the control plant. Analysis of enzymatic activity using hybridization with anti-chitinase antibody indicated that the level of chitinase activity in the transgenic tobacco carrying the enhancer is higher than that without enhancer. These data suggest that the enhancer improved the expression of chi transgene in tobacco.

Overexpression of chitinase gene with a GC-rich synthetic enhancer in tobacco plant (Nicotiana tabacum L.) Overekspresi gen kitinase dengan enhancer sintetis kaya GC pada tanaman tembakau (Nicotiana tabacum L.)

Ringkasan Perakitan tanaman perkebunan toleran terhadap serangan cendawan patogenik dilakukan dengan mengoverekspresikan gen penyandi kitinase. Untuk itu elemen DNA peningkat ekspresi (enhancer E52) yang berupa oligonukleotida 52 pb dan kaya kandungan basa purin (GC) disisipkan di ujung 5’ konstruk 35S-chi. Penyisipan E52 tersebut dilakukan secara lebih terarah pada situs ganda HindIII-SalI dari MCS pCAMBIA2301. Melalui situs HindIII yang terletak tepat di ujung 3’ E52, konstruk 35S-chi kemudian disambungkan dengan E52 pada pCAMBIA tersebut. Transformasi DNA rekombinan ke dalam sel tembakau dikerjakan melalui perantaraan Agrobacterium tumefaciens LBA4404. Sel tanaman transgenik diseleksi dan diregenrasi dalam media yang mengandung 3% sukrosa, 0,5 mg/L diregenerasikan pada media MS padat benzilaminopurin (BAP) dan 50 mg/L kanamisin. Pada media ini tunas transgenik tembakau mulai terbentuk setelah 5 minggu penanaman. Analisis tingkat aktivitas enzimatis menunjukkan bahwa aktivitas kitinase pada tembakau transgenik 40 hingga 80 kali lebih tinggi daripada non-transgenik. Pengujian hibridisasi protein menggunakan antibodi anti-kitinase, dot blot dan western blot, membuktikan bahwa enhancer tersebut dapat meningkatkan ekspresi transgen chi pada tanaman tembakau.Summary Development of estate crops tolerant to pathogenic fungi is conducted by overexpressing chi gene. For this purpose, a synthetic enhancer consisting of 52 base pairs and GC-rich was inserted at immediate 5’ end of a 35S-chi cassette. Insertion of the E52 was directed at HindIII-SalI restriction sites of the pCAMBIA2301 MCS. With HindIII restriction site located just after the 3’ end of the E52 sequence, the 35S-chi construct was then ligated with the E52 of the pCAMBIA. Transformation of the resulting recombinant DNA into tobacco cells was mediated by Agrobacterium tumefaciens LBA4404. The transgenic cells were selected and regenerated on a solid MS medium supplemented with 3% sucrose, 0.5 mg/L benzylamino purine (BAP) and 50 mg/L kanamycin. Tobacco shoots were initiated after 5 weeks inoculation on the selection media. Enzymatic analysis demonstrated that chitinase activity of transgenic tobacco was 40 to 80 folds higher than that of the control plant. Analysis of enzymatic activity using hybridization with anti-chitinase antibody indicated that the level of chitinase activity in the transgenic tobacco carrying the enhancer is higher than that without enhancer. These data suggest that the enhancer improved the expression of chi transgene in tobacco.

240 PRECISE GENOME EDITING OF PDX1 BY DIRECT INJECTION OF TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASES (TALENS) INTO PARTHENOGENETIC PIG EMBRYOS

Pancreatic and duodenal homeobox 1 (PDX1) is one of the transcription factors involved in pancreatic organogenesis and plays a critical role as an early lineage marker of pancreatic specification and β-cell differentiation. In mature pancreas, PDX1 regulates a large number of genes involved in maintaining β-cell identity and function. In mice and humans, its homozygous disruption results in pancreas agenesis, while heterozygous mutations have been associated with early-onset (MODY) and late-onset forms of Type II diabetes mellitus in humans. Knockout of the PDX1 gene in pigs may lead to the generation of an apancreatic phenotype, which in turn could allow the potential generation of an exogenic pancreas. Moreover, this could help to create a large animal model for human diabetes. We used transcription activator-like effector nucleases (TALEN) technology with the aim of studying the efficiency of precise editing by homologous recombination in parthenogenetically activated porcine embryos. Mature oocytes were activated by incubation in ionomycin (10 µM) for 20 min, followed by a 4-h incubation in DMAP (2 mM) + cytochalasin B (7.5 µg mL–1). Cytoplasmic injection was performed 14 h post-activation. Post injections, embryos were cultured for 6 days in NCSU23+BSA media at 38.5°C in a 5% CO2 atmosphere. We first evaluated the activity of a pair of TALENs targeting the functional domain of the PDX1 gene. Three concentrations of mRNA were microinjected (10, 20, and 40 ng µL–1) and blastocysts were analysed for non-homologous end-joining (NHEJ) by sequencing. The efficiency of indel mutations at the PDX1-target loci (either monoallelic or biallelic) was 34.5, 52.6, and 80.0% for the 10, 20, and 40 ng µL–1 concentrations, respectively. Next, we tested whether ssODNs (single-stranded oligodeoxynucleotide) coinjected with TALENs would permit precise homology direct repair (HDR) in porcine parthenotes. TALEN mRNA (40 ng µL–1) was coinjected with an ssODN donor template (50 ng µL–1) designed to incorporate a novel stop codon, HindIII restriction site, and a frame shift mutation. Cleavage and blastocyst rates were recorded at Days 2 and 6 of development, respectively, in the TALEN/ssODN injected group (n = 260 zygotes), buffer-injected embryos (n = 135 zygotes), and the non-injected group (n = 132 zygotes). Day 7 embryos were analysed for NHEJ and HDR by RFLP assay and Sanger sequencing after whole-genome amplification and PCR. Blastocyst rates were 15% (TALEN/ssODN-injected group), 27% (buffer-injected group), and 34% (non-injected group). A total of 30 blastocysts were analysed for HDR after whole-genome amplification. The majority of analysed blastocysts (28/30, 93%) were mutant. Among them, 10 (36%) incorporated the ssODN, from which 3 (30%) showed a KO genotype with a precise biallelic modification. We report here a highly efficient and precise TALEN-mediated gene knockout in swine embryos, which represents an alternative to cloning for phenotype evaluation of knockouts related to organogenesis.

245 EFFECTS OF Gyr (BOS TAURUS INDICUS) DONOR MITOCHONDRIAL DNA AND OVUM PICKUP ORDER ON IN VITRO EMBRYO PRODUCTION

Ovum pick (OPU) up is a technique upon which in vitro embryo production (IVP) depends. It permits donor cow maternal ancestry to be assessed by mtDNA analysis. Repetitive ablation of follicles is thought to interfere with the donor cow endocrine profile and influence embryo yield. The objective was to evaluate the effects of mtDNA and OPU order on IVP fertility traits. Gyr donors (85) were submitted to 363 OPU sessions (5 OPU sessions/donor). Donor mtDNA was extracted from leukocytes and sorted by the presence of the HindIII restriction site within the amplified region, indicative of Bos taurus taurus mtDNA (Paneto et al. 2008 Genet. Mol. Res. 7, 592). All animals in the donor pedigree were classified, and the population was divided into two groups according to their maternal genetic grouping: Bos taurus indicus and Bos taurus taurus. For statistical analyses, data from 5 OPU sessions per each donor were submitted to the mixed model procedure of SAS® (SAS Institute Inc., Cary, NC, USA), using the lowest Akaike value to determine the best covariance structure between repeated OPU session results. The model included the effect of OPU session order (1–5) and mtDNA-based maternal grouping (Bos taurus v. Bos indicus) as independent variables. Total and viable oocytes, as well as blastocyst yield per OPU session, were the dependent variables studied. The means of total and viable oocytes and blastocysts produced per donor per OPU were compared by the Tukey test, at 5% significance. The combined OPU sessions resulted in 6084 oocytes, 2537 embryos, which produced 1105 pregnancies. Mean numbers (n = 42 donors/OPU session) of total and viable oocytes between OPU sessions 1 (31.9 ± 4.6 and 19.2 ± 2.9), 2 (35.3 ± 3.5 and 21.2 ± 2.2), 3 (28.9 ± 3.7 and 19.3 ± 2.4), 4 (25.0 ± 5.2 and 18.9 ± 33), and 5 (20.5 ± 6.3 and 13.8 ± 4.0) did not differ. Mean blastocyst production after IVP between OPU sessions 1 (6.5 ± 1.4), 2 (5.15 ± 1.04), 3 (5.5 ± 1.1), 4 (5.8 ± 1.6), and 5 (5.2 ± 1.9) was similar. Mean viable oocyte number was greater (P < 0.0001) for B. t. taurus (21.7 ± 1.4, n = 192 OPU sessions) compared with B. t. indicus (16.1 ± 0.9, n = 138 OPU sessions) maternal genetic groupings. However, in vitro blastocyst yields were similar (P = 0.23) between maternal genetic groupings (7.3 ± 1.6 and 6.2 ± 1.4 for B. taurus and B. indicus, respectively). In conclusion, repeated OPU sessions did not reduce oocyte and embryo yields as expected. Maternal B. taurus genetic origin was associated with higher oocyte quality, although it was not translated into higher embryo yields after in vitro culture. Results warrant further research, which may result in additional selection criteria for OPU Gyr donors considering their maternal genetic background.

“Red Cell Complement Loading In PNH Patients On Eculizumab Is Associated With a C3 Polymorphism Which Influences C3 Function, Predicts For Increased Extravascular Hemolysis and Provides a Rationale For C3 Inhibition.”

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired bone marrow disorder characterised by intravascular hemolysis and hemoglobinuria, potentially life-threatening thrombosis and an association with aplastic anemia. Most of the clinical features and complications of PNH are due to the unopposed activity of complement due to the absence of CD59 and CD55, two key regulators of complement. Eculizumab prevents the cleavage of C5 complement thereby preventing terminal complement activity and protecting PNH cells from lysis. The inhibition of C5 preserves the early part of complement pathway and leads to the build up of C3 on the PNH red cells, perhaps in part due to their lack of CD55. The majority of PNH patients receiving eculizumab have evidence of extravascular haemolysis that can be clinically significant, including with anemia, hyperbilirubinemia and in some a continued requirement for transfusions. This extravascular hemolysis in thought to be due to the C3 loading of PNH red cells. Methods We report the C3-loading of the PNH red cells from 119 patients treated with eculizumab and correlate this with hemoglobin, LDH, bilirubin, reticulocytes and transfusions. We have studied genetic polymorphisms that affect both C3 and FCγR. We have genotyped 46 eculizumab patients for a functional mutation in the C3 gene (rs2230199). The two alleles of this gene can be distinguished by the presence or absence of a HindIII restriction site that distinguishes the electophoretically slow (arg80) from the electrophoretically fast (gly80) allotype. The fast (C3F) allotype allele of this snp is associated with a range of disorders including age-related macular degeneration, IgA nephropathy, systemic vasculitis and partial lipodystrophy. APL-1 is a small cyclic peptide that binds to and inhibits the activation of complement C3. APL-2 is a large conjugate of APL-1 with enhanced bioactivity and a long systemic half-life. APL-1 and APL-2 molecules as well as other complement inhibitors were studied for lysis of red cells and C3 loading in vitro in a modified Ham test in which flow cytometry was used to identify non-lysed cells. Results Out of the 119 Eculizumab treated patients, 55 (46.2%) required at least one transfusion on treatment. 110 patients had C3 detectable by flow cytometry on their PNH red cells (mean of 19.8%; range: <0.1 to 64.6%). C3-loading was not seen on the normal red cells from the same patients on treatment nor on the PNH red cells in patients not receiving eculizumab. The mean LDH (735IU/l) and reticulocyte count (193 x 109/l) were not statistically significantly different for the transfused group compared to the non-transfused group (518 and 163 respectively). Mean PNH C3 and RBC C3 did not differ stastistically between the transfused and non-transfused groups (26.20 Vs 24.78 PNH C3;15.96 vs 15.09 RBC C3 respectively). We studied one functional polymorphism in the Fcγ receptor but this showed no correlation with haemolytic parameters. Conversely, for the C3 polymorphism eculizumab-treated patients homozygous for the slow (C3S) allele had a significantly higher degree of C3 loading on PNH red blood cells with C3S/C3S having a mean of 33.7% C3 loaded PNH red cells (n=26), C3S/C3F 19.0% (n=19) and C3F/C3F 12.8% (n=3)(all P<0.01). Homozygote C3S also had increased reticulocytes (P<0.01) and bilirubin (P<0.01). The C3S allele has previously been shown to be more efficient in a haemolysis assay using sheep erythrocytes. This polymorphism appears to explain much of the variation in C3 loading between different patients with PNH. In vitro experiments show that inhibitors of C5, such as eculizumab, protect the PNH red cells from lysis but lead to a very rapid deposition of C3 on the PNH red cells. However both APL1 and APL2 demonstrated similar protection of PNH red cell lysis with virtually no C3 loading on PNH red cells. Conclusion A significant proportion of patients on eculizumab experience extravascular haemolysis with the majority of patients developing C3 loading. We show for the first time that a functional polymorphism in the C3 gene is associated parameters of hemolysis. The S(low) allele alters the function of C3 protein and appears to be associated with extravascular haemolysis in patients with PNH. The C3 inhibitors, APL-1 and APL-2, protect PNH red cells and prevent C3 loading in vitro and if safe to be given chronically would be expected to reduce extravascular hemolysis significantly. Disclosures: Hill: Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Kelly:Alexion: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Hillmen:Alexion: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Definition of interferon gamma-response elements in a novel human Fc gamma receptor gene (Fc gamma RIb) and characterization of the gene structure.

The human Fc gamma RI (CD64) is a high affinity receptor for the Fc portion of immunoglobulin (Ig), and its constitutively low expression on the cell surface of monocyte/macrophage and neutrophils is selectively upregulated by interferon gamma (IFN-gamma) treatment (Perussia, B., E. T. Dayton, R. Lazarus, V. Fanning, and G. Trinchieri. 1983. J. Exp. Med. 158:1092). Three distinct cDNAs have been cloned and code for proteins that predict three extracellular Ig-like domains (Allen, J.M., and B. Seed. 1989. Science [Wash. DC]. 243:378). Several differences in the coding region of these cDNAs suggest that in addition to polymorphic differences a second Fc gamma RI gene could possibly exist. This alternative Fc gamma RI gene (Fc gamma RIb) was defined by the lack of a genomic HindIII restriction site (van der Winkel, J. G. J., L. U. Ernst, C. L. Anderson, and I. M. Chiu. 1991. J. Biol. Chem. 266:13449). We describe the characterization a second gene (Fc gamma RIb) that has a termination codon in the third extracellular domain and therefore predicts a soluble form of a termination codon in the third extracellular domain and therefore predicts a soluble form of the receptor. We also define two distinct IFN-gamma-responsive regions in the 5' flanking sequence of Fc gamma RIb that resemble motifs that have been defined in the class II major histocompatibility complex promoter. The Fc gamma RIb promoter does not possess canonical TATA or CCAAT boxes, but does possess a palindromic motif that closely resembles the initiator sequence identified in the terminal deoxynucleotidyl transferase/human leukocyte IFN/adeno-associated virus type II P5 gene promoters (Smale, S. T., and D. Baltimore. 1989. Cell. 57:103; Seto, E., Y. Shi, and T. Shenk. 1991. Nature [Lond.]. 354:241; Roy, A. L., M. Meisterernst, P. Pognonec, and R. C. Roeder. 1991. Nature [Lond.]. 354:245) virus type II P5 gene promoters raising interesting questions as to its role in the basal and myeloid-specific transcription of this gene.

Bipartite structure of the 5S ribosomal gene family in a Drosophila melanogaster strain, and its evolutionary implications.

Knowledge of multigenic family organization should provide insight into their mode of evolution. Accordingly, we characterized the 5S ribosomal gene family in the Drosophila melanogaster strain ry506. The 5S genes in this strain display a striking HindIII restriction difference compared to the "standard" D. melanogaster 5S genes. The sequence of three ry506 5S genes was determined. We show that the HindIII restriction site heterogeneity within the ry506 5S family most probably results from the same point mutation, suggesting that a single 5S variant was propagated into the 5S cluster of this strain. Furthermore, we demonstrate that the structural organization of the 5S genes in ry506 is a bipartite structure, i.e., that about 40% of the 5S genes constitute a HindIII+/HindIII- mixed cluster, while those remaining constitute an homogeneous HindIII- cluster. The events which might lead to such an heterogeneous pattern are discussed from an evolutionary point of view.

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Sequence and expression of the dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae.

The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).

Tc1(Hin): a form of the transposable element Tc1 in Caenorhabditis elegans

In this paper we describe the coexistence of two forms of the transposable element Tc1 in the genome of the nematode Caenorhabditis elegans. A copy of the variant form has been isolated from the Bergerac genome and characterized. Restriction mapping and DNA sequencing have shown that a G to T transversion generated a HindIII restriction site to form the variant Tc1(Hin). The presence of this new restriction site makes this variant easily detectable on genomic blot hybridizations. There are approximately 20 copies of Tc1(Hin) amongst the Tc1's present in the Bergerac genome. Bergerac has approximately 250 copies of Tc1 per genome, whereas Bristol has about 30. In the Bristol strain we detected at least one copy Tc1(Hin). The ratio of Tc1 (Hin) to total Tc1's is similar in the genomes of Bristol and Bergerac, even though they have markedly different total numbers of Tc1. Our results suggest that a trans-acting change in either the elements or the host genome was responsible for the expansion of Tc1 copy number in the Bergerac genome.