scholarly journals Molecular characterization and n use efficiency of LeAlaAT ‘Mekongga’ transgenic rice lines

2021 ◽  
Vol 53 (4) ◽  
pp. 723-736
Author(s):  
D.S. Yulita ◽  
B.S. Purwoko ◽  
A. Sisharmini ◽  
A. Apriana ◽  
T.J. Santoso ◽  
...  
Keyword(s):  
Southern Blot ◽  
Molecular Characterization ◽  
Transgenic Rice ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Marker Gene ◽  
Direct Pcr ◽  
Transgenic Lines ◽  
Use Efficiency ◽  
N Use

Genetic engineering is one of the strategies for developing nitrogen (N)-use-efficient rice (Oryza sativa) varieties. One gene that plays an indirect role in N metabolism is alanine aminotransferase (AlaAT). It can efficiently increase N content and crop yield. In a previous study, the tomato AlaAT gene (LeAlaAT) was successfully isolated and introduced into ‘Mekongga’ rice. The present research was conducted during 2018 and 2019 at the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Bogor, Indonesia. The objectives of the present study were to perform the molecular characterization of LeAlaAT ‘Mekongga’ rice lines on the basis of the hpt marker gene, the direct PCR of the LeAlaAT fragment, and the phenotypic evaluation of the selected LeAlaAT T1 ‘Mekongga’ rice lines in response to different N fertilizer rates (0 kg ha−1 [control] and 60, 90, and 120 kg ha−1). This research involved three activities, namely (1) Southern blot analysis, (2) direct PCR, and (3) N use efficiency (NUE) test of ‘Mekongga’ transgenic lines. Southern blot analysis revealed that in T0 transgenic lines, the copy number of the hpt marker gene varied from 1 to 3. Direct PCR confirmed the presence of the AlaAT fragment in the T1 generation of five ‘Mekongga’ transgenic lines. The five transgenic lines showed high panicle number, biomass weight, shoot dry weight, and total grain weight under 120 kg ha−1 nitrogen. The high agronomical NUE of transgenic lines under 120 kg ha−1 N implied that the transgenic rice lines have the potential for efficient N use at a certain minimum level of N (120 kg ha−1 of nitrogen) and should be further evaluated at high N levels.

scholarly journals Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2566-2571 ◽  
Author(s):  
FM Stewart ◽  
RB Crittenden ◽  
PA Lowry ◽  
S Pearson-White ◽  
PJ Quesenberry
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Normal Male ◽  
Normal Donor ◽  
Long Term Stability ◽  
Donor Bone Marrow ◽  
Male Donor ◽  
Marrow Cells

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

scholarly journals Mutational analysis of the CDKN2 (MTS1/p16ink4A) gene in primary B-cell lymphomas

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2724-2731 ◽  
Author(s):  
T Uchida ◽  
T Watanabe ◽  
T Kinoshita ◽  
T Murate ◽  
H Saito ◽  
...  
Keyword(s):  
B Cell ◽  
Missense Mutation ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Lymphocytic Leukemia ◽  
Sscp Analysis ◽  
Putative Tumor Suppressor Gene

Abstract The CDKN2 gene located on chromosome 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16. This gene is a putative tumor-suppressor gene because of its frequent alterations in many kinds of tumor cell lines. We analyzed the CDKN2 gene to evaluate its alterations in 52 primary specimens of non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) of B-cell origin by Southern blot analysis, polymerase chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing. By Southern blot analysis, we showed homozygous deletion of the CDKN2 gene in 3 of 42 patients with B-NHL (7.1%). After screening by PCR-SSCP analysis, direct sequencing identified one missense mutation at codon 72 (nucleotide 233) and two frameshifts due to a 35-bp deletion arising at codon 49 (nucleotides 163 to 175) in patients with B-NHL (3 of 42, 7.1%). In the patient carrying the missense mutation, hemizygous deletion of the CDKN2 gene was also suspected. In this study, we detected alterations in CDKN2 in 6 of 42 patients (14.3%) with B-NHL and in none of 10 patients with B-CLL. Our results suggest that the CDKN2 alterations contribute in tumorigenesis in some patients with B-NHL.

Analysis of HLA-B35 variants andB35 haplotypes by isoelectric focusing and Southern blot analysis

1989 ◽  
Vol 30 (1) ◽  
pp. 63-65 ◽  
Author(s):  
Erich Lederer ◽  
Elfriede Nößner ◽  
Rudolf Wank ◽  
Dolores J. Schendel
Keyword(s):  
Southern Blot ◽  
Isoelectric Focusing ◽  
Blot Analysis ◽  
Southern Blot Analysis

scholarly journals Retroviral vector system for the study of cDNA gene formation.

1988 ◽  
Vol 8 (6) ◽  
pp. 2328-2334 ◽  
Author(s):  
R Dornburg ◽  
H M Temin
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Retroviral Vector ◽  
Vector System ◽  
Cis Acting ◽  
Infected Cells ◽  
Retroviral Replication

A retroviral vector system was developed to study the retrotransposition of RNAs lacking all cis-acting sequences required for normal retroviral replication. Our experiments indicate that such RNAs can be encapsidated in retroviral proteins, reverse transcribed, and integrated to form functional cDNA genes in infected cells. The frequency of this process, however, was approximately 8 orders of magnitude less than that of normal retroviral replication. The efficiency was limited at each step in this process. Investigation of seven cDNA genes by Southern blot analysis revealed that all of them were truncated at either the 3' or the 5' end or both. These truncations are not seen with natural cDNA genes and raise the question of retroviral involvement in their formation.

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