Construction of yeast one-hybrid library and screening of transcription factors regulating LhMYBSPLATTER expression in Asiatic hybrid lilies (Lilium spp.)

Background Anthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TF) LhMYBSPLATTER (formerly known as LhMYB12-Lat), identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in ‘Tiny Padhye’ of Tango Series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYBSPLATTER, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYBSPLATTER in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized. Results In this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYBSPLATTER gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400 to 2000 bp, and the library capacity reached 1.6 × 106 CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYBSPLATTER promoter. Among them, ERFs, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium. Conclusion A yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYBSPLATTER were screened, which may provide a theoretical basis for the study of floral pigmentation.

Construction of Yeast One-hybrid Library and Screening of Transcription Factors Regulating LhMYB12-Lat Expression in Asiatic Hybrid Lilies (Lilium spp.)

Background: Anthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TFs) LhMYB12-Lat, identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in ‘Tiny Padhye’ of Tango series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYB12-Lat, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYB12-Lat in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized. Results: In this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYB12-Lat gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400-2000 bp, and the library capacity reached 1.6 × 106 CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYB12-Lat promoter. Among them, ERF, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium.Conclusion: A yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYB12-Lat were screened, which may provide a theoretical basis for the study of floral pigmentation.

cDNA Cloning of a Bovine Insulin-like growth factor-1 from Egyptian Buffalos and Expression of its Recombinant Protein in Escherichia coli

ABSTRACT Insulin-like growth factor-1 (IGF-1) is regarded as a crucial clinically significant therapeutic agent against several pathological conditions. Recently, recombinant DNA (rDNA) technology has enabled the production of many drugs of rDNA-origin including IGF-1. Securing a readily available supply of IGF-1 is invaluable to clinical research and biotechnological domains. In this work, the cloning of a full-length bovine IGF-1 cDNA and the successful expression of its cognate recombinant IGF-1 protein is reported. Single-strand cDNA was prepared from liver tissues, through the specific reverse transcription (RT) of IGF-1 mRNA. Subsequently, a PCR amplicon of ~543bp was successfully amplified. Recombinant pTARGET™ vector harboring IGF-1 insert was successfully cloned into competent E. coli JM109 cells. SDS-PAGE analysis revealed that the recombinant IGF-1 has been expressed at the expected size of 7.6kDa. The outcome provides a robust basis for transecting the recombinant pTARGETTM vector, harboring the IGF-1 cDNA insert, into mammalian cells. Optimal initial glucose concentration was found to be 10g/l with corresponding protein concentration of 6.2g/l. The proliferative biological activity crude recombinant IGF-1 protein was verified on HeLa cell lines. This is envisaged to facilitate large-scale production of recombinant IGF-1 protein, thereby enabling thorough investigation of its clinical and pharmaceutical effects.

Konstruksi pustaka cDNA dari daun klon karet AVROS 2037 yang diinfeksi patogen Corynespora cassiicola Construction of a cDNA library from leaf of AVROS 2037 rubber clone infected by Corynespora cassiicola pathogen

SummaryConstruction of cDNA library derived fromtranscripts made under certain condition is animportant first step to understand diseaseresistant mechanisms. To identify rubber genesor transcripts involved in defense responsetoward Corynespora cassiicola, cDNA librarywas constructed using rubber clone AVROS2037, one of resistant clone to this pathogen.cDNA library was constructed based on thestrategy of leaves infection using conidia, withthe assumption that transcript expression relatedto defense response would be induced bypathogen infection. RNA was isolated from leavesthree days after inoculation with conidia ofC. cassiicola. Steps involved in the cDNA libraryconstruction were RNA isolation, mRNApurification, cDNA synthesis, vector modifcation,cDNA insert ligation, plasmid transformation andclone verifications. Each gram of leaf producedapproximately 300  g RNA, and 0.25% of themwas mRNA. The mRNA was used to synthesizedcDNA. Ligation of cDNA and modified vectorwas facilitated by restriction enzyme SfiI. Theconstructs were transformed into the E. coliDH5 competent cells. A total of 8000 colonieswere produced. Random examination of 270colonies showed that approximately 93% of thesecolonies carried plasmid vector with DNA insertsize of 200 – 2000 bp, with average size of 500 –800 bp. cDNA library construction of rubberleaves from AVROS 2037 clone as well as somenecessary modification steps are presented in thispaper.RingkasanKonstruksi pustaka cDNA yang me-ngandung transkrip yang diekspresikan dalamkondisi tertentu merupakan tahap awal yangsangat penting dalam berbagai studi biologi.Untuk mengidentifikasi gen karet atau transkripyang berperan dalam respons pertahanan tanamankaret terhadap Corynespora cassiicola, pustakacDNA dibuat dengan menggunakan daun klonAVROS 2037 yang merupakan salah satu klonresisten terhadap patogen tersebut. PustakacDNA dibuat berdasarkan strategi menginfeksidaun dengan konidia C. cassiicola denganpertimbangan bahwa ekspresi transkrip yangberperan dalam respons pertahanan akandiinduksi oleh adanya infeksi patogen. Dengandemikian pustaka cDNA yang dibuat diharapkanmengandung gen atau bagian gen yang ber-hubungan dengan respons pertahanan. RNAdiisolasi dari daun setelah daun diinokulasiselama tiga hari dengan konidia C. cassiicola.Beberapa tahapan telah dilakukan, dimulaidengan isolasi RNA, pemurnian mRNA, sintesiscDNA, modifikasi vektor kloning, ligasi fragmencDNA utas ganda dengan vektor kloning sertatransformasi hasil ligasi ke bakteri Escherichiacoli DH5 kompeten. Dari setiap gram jaringandaun berhasil diisolasi RNA sekitar 300 g, dandari jumlah tersebut sekitar 0,25% mRNA dapatdiisolasi. mRNA yang diisolasi digunakan untuksintesis cDNA. cDNA dipotong dengan enzimrestriksi SfiI dan diligasi ke vektor plasmid yangdimodifikasi dengan menyisipkan situs enzimSfiI. cDNA-vektor rekombinan ditransformasi kedalam sel bakteri E. coli DH5 kompeten meng-gunakan metode standar. Transformasi konstrukini menghasilkan 8.000 koloni. Pengujian PCRterhadap 270 koloni yang dipilih secara acakmengindikasikan bahwa sekitar 93% kolonitersebut membawa cDNA sisipan dengan ukuranfragmen cDNA yang menyisip berkisar antara200 sampai 2000 bp. cDNA sisipan terbanyakterdapat pada ukuran antara 500 – 800 bp. Dalamtulisan ini dibahas tahap demi tahap proses yangdilakukan untuk membuat pustaka cDNA asaldaun karet klon AVROS 2037 serta beberapamodifikasi yang diperlukan.

Konstruksi pustaka cDNA dari daun klon karet AVROS 2037 yang diinfeksi patogen Corynespora cassiicola Construction of a cDNA library from leaf of AVROS 2037 rubber clone infected by Corynespora cassiicola pathogen

SummaryConstruction of cDNA library derived fromtranscripts made under certain condition is animportant first step to understand diseaseresistant mechanisms. To identify rubber genesor transcripts involved in defense responsetoward Corynespora cassiicola, cDNA librarywas constructed using rubber clone AVROS2037, one of resistant clone to this pathogen.cDNA library was constructed based on thestrategy of leaves infection using conidia, withthe assumption that transcript expression relatedto defense response would be induced bypathogen infection. RNA was isolated from leavesthree days after inoculation with conidia ofC. cassiicola. Steps involved in the cDNA libraryconstruction were RNA isolation, mRNApurification, cDNA synthesis, vector modifcation,cDNA insert ligation, plasmid transformation andclone verifications. Each gram of leaf producedapproximately 300  g RNA, and 0.25% of themwas mRNA. The mRNA was used to synthesizedcDNA. Ligation of cDNA and modified vectorwas facilitated by restriction enzyme SfiI. Theconstructs were transformed into the E. coliDH5 competent cells. A total of 8000 colonieswere produced. Random examination of 270colonies showed that approximately 93% of thesecolonies carried plasmid vector with DNA insertsize of 200 – 2000 bp, with average size of 500 –800 bp. cDNA library construction of rubberleaves from AVROS 2037 clone as well as somenecessary modification steps are presented in thispaper.RingkasanKonstruksi pustaka cDNA yang me-ngandung transkrip yang diekspresikan dalamkondisi tertentu merupakan tahap awal yangsangat penting dalam berbagai studi biologi.Untuk mengidentifikasi gen karet atau transkripyang berperan dalam respons pertahanan tanamankaret terhadap Corynespora cassiicola, pustakacDNA dibuat dengan menggunakan daun klonAVROS 2037 yang merupakan salah satu klonresisten terhadap patogen tersebut. PustakacDNA dibuat berdasarkan strategi menginfeksidaun dengan konidia C. cassiicola denganpertimbangan bahwa ekspresi transkrip yangberperan dalam respons pertahanan akandiinduksi oleh adanya infeksi patogen. Dengandemikian pustaka cDNA yang dibuat diharapkanmengandung gen atau bagian gen yang ber-hubungan dengan respons pertahanan. RNAdiisolasi dari daun setelah daun diinokulasiselama tiga hari dengan konidia C. cassiicola.Beberapa tahapan telah dilakukan, dimulaidengan isolasi RNA, pemurnian mRNA, sintesiscDNA, modifikasi vektor kloning, ligasi fragmencDNA utas ganda dengan vektor kloning sertatransformasi hasil ligasi ke bakteri Escherichiacoli DH5 kompeten. Dari setiap gram jaringandaun berhasil diisolasi RNA sekitar 300 g, dandari jumlah tersebut sekitar 0,25% mRNA dapatdiisolasi. mRNA yang diisolasi digunakan untuksintesis cDNA. cDNA dipotong dengan enzimrestriksi SfiI dan diligasi ke vektor plasmid yangdimodifikasi dengan menyisipkan situs enzimSfiI. cDNA-vektor rekombinan ditransformasi kedalam sel bakteri E. coli DH5 kompeten meng-gunakan metode standar. Transformasi konstrukini menghasilkan 8.000 koloni. Pengujian PCRterhadap 270 koloni yang dipilih secara acakmengindikasikan bahwa sekitar 93% kolonitersebut membawa cDNA sisipan dengan ukuranfragmen cDNA yang menyisip berkisar antara200 sampai 2000 bp. cDNA sisipan terbanyakterdapat pada ukuran antara 500 – 800 bp. Dalamtulisan ini dibahas tahap demi tahap proses yangdilakukan untuk membuat pustaka cDNA asaldaun karet klon AVROS 2037 serta beberapamodifikasi yang diperlukan.

An improved protocol for small RNA library construction using High Definition adapters

Next generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with greatly reduced proportion of adapter-adapter products due to the degradation of nonligated 3’ adapters. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue.

Adenovirus-interferon γ (TG1042) demonstrates good tolerance and efficacy in relapsing primary cutaneous lymphoma (PCL): Final results of a phase I/II multicentric trial

7539 Background: Interferons (IFNs), which can offset the Th2 dominance associated with PCL have been successfully used to treat these PCL. Intratumoral (i.t.) injection of TG1042 (a non-replicating recombinant adenovirus with a human IFNγ cDNA insert) induces high local production of IFNγ without severe toxicity associated with systemic delivery. Methods: We undertook a phase I/II multicentric trial of repeated, i.t. injections of TG1042 in patients with advanced primary T cell (CTCL) or B cell (CBCL) CL. One to 3 lesions were injected on day 1, 8, and 15 (a 4-week cycle) and thereafter up to 12 cycles. Immunohistochemistry and quantitative PCR were performed on injected lesions biopsied at baseline and after the 1st cycle. In the phase I, 18 patients were enrolled in 3 successive cohorts at the doses of 3 × 109 viral particles (vp) (n = 3), 3 × 1010 vp (n = 3) and 3 × 1011 vp (n = 12). In the phase II, 18 evaluable patients were planned to be treated at 3 × 1011 vp. Results: To date, enrollment is complete, 39 patients (32 CTCL and 7 CBCL) have been included, 9 of them are still on treatment. Patients received a median of 4 lines of prior therapy. 11 patients were at stage Ib and 16 patients at higher stage. Altogether, 245 injections of TG1042 have been administered. Treatment was well tolerated with 8 grade 3 related adverse events. Injection site reaction and flu like syndrome are the most common adverse events. Histology demonstrates pronounced changes in infiltrate patterns with signs of vasculitis, increased numbers of eosinophils, neutrophils, CD8 and TIA-1+ve cells. CD4/CD8 ratio decreased in most tumors. Transgene-IFNγ mRNA was detected in injected lesions. Gene expression analysis of biopsies and PBMC shows up-regulation of IFNγ genes. Local clinical response has been observed in 17 (including 9 complete responses [CR]) out of 31 evaluable patients. 13 global responses (7 CR) out of 30 evaluable patients have been observed. All 5 evaluable CBCL responded (3 CR). Conclusions: These results demonstrate that TG1042 is well tolerated and presents a potential significant benefit for the treatment of both CTCL and CBCL. A phase II is planned in patients with CBCL to confirm those encouraging results. [Table: see text]

Adenovirus-Interferon-g (TG1042) Demonstates Clinical Activity in Relapsing Primary Cutaneous Lymphoma (PCL): A Phase I/II Multicentric Trial.

PCL has been successfully treated with interferons (IFNs), which can offset the Th2 dominance associated with PCL. Intratumoral (IT) injection of TG1042 (a non-replicating recombinant adenovirus with a human IFN-γ cDNA insert) allows high local levels of IFN-γ without severe toxicity induced by systemic delivery. We undertook a multicentric, phase I/II, open-label, trial of repeated, IT injection of TG1042 in patients with advanced primary T cell (CTCL) and multilesional B cell (CBCL) cutaneous lymphoma. The designated lesions were injected on day 1, 8 and 15 with no injection in the fourth week (1 cycle) and thereafter up to 4 cycles. Immunohistochemistry and qPCR were performed on injected lesions biopsied at baseline and after each cycle. In the phase I step, undertaken on a monocentric basis, 9 patients were enrolled in 3 successive cohorts at the doses of 3×109 viral particles (vp), 3×1010 and 3×1011 vp injected in only one lesion and 9 additonal patients were treated at the doses of 3×1011 vp divided in up to 3 lesions. In the phase II step, 18 additional patients have been planned to be treated on a multicentric basis at 3×1011 vp injected in up to 3 lesions. To date, the 36 planned patients (30 CTCL and 6 CBCL) have been enrolled and received a total of 280 injections, 26 patients have completed the treatment. Treatment is well tolerated with only 3 grade III toxicity. Injection site reaction and flue like syndrom are the most commonly observed adverse events. Local clinical response has been observed in 13 (7 complete responses) out of 27 evaluable patients. Disappearance of non-injected lesions was also observed and led to 12 overall responses (6 complete responses) out of 26 evaluable patients. Histology demonstrates pronounced differences in infiltrate patterns following treatment, with signs of vasculitis and increased numbers of eosinophils, neutrophils, CD8 and TIA-1+ve cells. CD4/CD8 ratio decreased in most tumors. Transgene-IFN-γ mRNA was detectable in injected lesions. Gene expression analysis of biopsies and PBMC shows up-regulation of IFN-γ and various immune response-associated genes. Final clinical and immunological data will be presented. In conclusion, these data show that the administration of TG1042 is well tolerated and results in clinical responses associated with in situ immunological changes. These encouraging results demonstrate a potential significant benefit of TG1042 for the treatment of primary cutaneous lymphoma.

Adenovirus-mediated intralesional interferon-γ gene transfer induces tumor regressions in cutaneous lymphomas

Primary cutaneous lymphomas have been successfully treated with interferons (IFNs), counterbalancing the T-helper 2 (Th2)-skewing state. We undertook a phase 1, open-label, dose-escalating trial of repeated intratumoral administration of TG1042 in patients with advanced primary cutaneous T-cell lymphomas (CTCLs) and multilesional cutaneous B-cell lymphomas (CBCLs). TG1042 is a third-generation, nonreplicating human adenovirus vector containing a human IFN-γ cDNA insert. Nine patients (7 CTCL, 2 CBCL) were enrolled at the following TG1042 doses: 3 × 109, 3 × 1010, and 3 × 1011 total particles. Local clinical response was observed in 5 of 9 treated patients (3 patients with complete response [CR] and 2 patients with partial response [PR]). Out of these, 3 patients showed systemic CR with the clearance of other noninjected skin lesions. Clinical response lasted for a median of 3 months (range, 1-6 months). Adverse events were mostly of grades 1 and 2. Seven of 9 treated patients had a detectable TG1042-derived IFN-γ message in injected lesions after the first treatment cycle. A TG1042-IFN-γ message was also detectable after several treatment cycles. We demonstrate the induction of humoral immune response to lymphoma tumor-antigen se70-2 after treatment. Our study shows that intralesional injections of TG1042 are both safe and well tolerated. (Blood. 2004;104:1631-1638)

Effect of alpha-1 antitrypsin Portland variant (α1-PDX) on HIV-1 replication

The present work investigated the potential role of alpha-1 antitrypsin Portland variant (α1-PDX), a bioengineered serine proteinase inhibitor (serpin), in the interference with the viral replication of HIV-1, induction of syncytia and maturation of envelope glycoprotein gp160 to gp120 and gp41. A Jurkat lymphoid cell line transfected with a plasmid containing the α1-PDX cDNA (J-PDX) and expressing the protein in a stable manner was infected with HIV-1Lai. Controls were Jurkat cells transfected with the same vector pcDNA3 without the cDNA insert (J-pcDNA3). The results showed that viral replication of HIV-1 was significantly inhibited with a delay in replication kinetics in J-PDX cells as compared with J-pcDNA3 cells. In addition, a comparison of the infectious capacity of viruses produced in the presence and absence of α1-PDX revealed that this capacity differed. It was found that α1-PDX exerts its effect by interfering with the formation of syncytia between J-PDX cells infected with gp160 recombinant vaccinia virus, or after infection by HIV-1 and co-culture with uninfected Molt-4 cells. In contrast, when the same experiments were performed with J-pcDNA3 cells, a large number of syncytia was obtained. Analysis of viral proteins by Western blotting and densitometry showed that the inhibition of the cytopathic effect of HIV-1 and viral replication was correlated with the capacity of α1-PDX to interfere with the maturation of gp160 to gp120 and gp41.