scholarly journals Application of Cleaved Amplified Polymorphic Sequence Method for Analysis of Cytoplasmic Genome among Aurantioideae Intergeneric Somatic Hybrids

2003 ◽  
Vol 128 (2) ◽  
pp. 225-230 ◽  
Author(s):  
Samia Lotfy ◽  
Francois Luro ◽  
Françoise Carreel ◽  
Yann Froelicher ◽  
Delphine Rist ◽  
...  
Keyword(s):  
Fragment Length Polymorphism ◽  
Somatic Hybrids ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Universal Primers ◽  
Genetic Studies ◽  
Cytoplasmic Genome ◽  
Cleaved Amplified Polymorphic Sequence ◽  
Pcr Rflp ◽  
Sequence Method

Somatic hybridization allows the creation of new patterns of nuclear, mitochondrial and chloroplastic association. It is therefore necessary to master cytoplasmic molecular markers to determine the genetic origin of both organelles of plantlets obtained from protoplasts fusion. In the case of Citrus and related genera, only southern blot hybridization and restriction fragment-length polymorphism (RFLP) techniques were used for this task until now. Here, we describe the use in the Aurantioideae subfamily, of a simple and non labeling cleaved amplified polymorphic sequence (CAPS) technique, to determine the cytoplasmic genome origin of intergeneric somatic hybrids. Mitochondrial and chloroplastic universal primers previously selected for population genetic studies in Quercus by Demesure et al. (1995) are used with some modifications. The variability of cytoplasmic genome among somatic fusion partners is detected by coupling amplification and restriction reactions. Digested DNA fragments are analyzed by agarose gel electrophoresis (PCR-RFLP). This technique has been applied for the analysis of the cytoplasmic constitution of somatic hybrids arising from intergeneric, intersubtribal and intertribal combinations. Systematic transmission of the mitochondria from protoplasts isolated from embryogenic callus parents was confirmed.

Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids

Genome ◽  
2007 ◽  
Vol 50 (5) ◽  
pp. 443-450 ◽  
Author(s):  
Marina Iovene ◽  
Salvatore Savarese ◽  
Teodoro Cardi ◽  
Luigi Frusciante ◽  
Nunzia Scotti ◽  
...  
Keyword(s):  
Parental Species ◽  
Somatic Hybrids ◽  
Solanum Bulbocastanum ◽  
Genome Composition ◽  
Specific Primers ◽  
Genetic Studies ◽  
Cytoplasmic Genome ◽  
Intergenic Regions

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.

scholarly journals Genotype and Parental Combination Influence Efficiency of Cybrid Induction in Citrus by Electrofusion

HortScience ◽  
1996 ◽  
Vol 31 (2) ◽  
pp. 275-278 ◽  
Author(s):  
Takaya Moriguchi ◽  
Tetsushi Hidaka ◽  
Mitsuo Omura ◽  
Toshiaki Motomura ◽  
Tomoya Akihama
Keyword(s):  
Somatic Hybrids ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Restriction Pattern ◽  
Specific Cell ◽  
Nuclear Rdna ◽  
Rough Lemon ◽  
Regenerated Plants ◽  
Parental Combination ◽  
Restriction Fragment Pattern

Interspecific hybridizations by electrofusion of embryogenic callus cells from `Seminole' tangelo (Citrus reticulata Blanco × C. paradisi Macf.), `Hazzara (Abohar)', or `Ohta' ponkan (C. reticulata Blanco) and leaf cells from `Lisbon' lemon [C. limon (L.) Burm. f.] or rough lemon (C. jambhili Lush.), respectively, were performed. Electrofusion of `Seminole' tangelo and `Lisbon' lemon, `Hazzara (Abohar)' and rough lemon, and `Ohta' ponkan and rough lemon resulted in 33, 43, and 36 plants, respectively. Seven to 10 plants in each combination were selected randomly and used to investigate nuclear and cytoplasmic genomes. Regenerated plants derived from electrofusion of `Seminole' tangelo and `Lisbon' lemon, and `Hazzara (Abohar)' and rough lemon possessed the same restriction fragment pattern for nuclear rDNA as that of the mesophyll parents and the same mitochondrial DNA (mtDNA) restriction pattern as that of the respective suspension parents, indicating that they were cybrids. In contrast, all the plants resulting from a combination between `Ohta' ponkan and rough lemon were confirmed by nuclear rDNA and mtDNA analysis to be somatic hybrids. The analysis of chromosome number supported the results of Southern blot hybridization. The results suggest that specific cell lines, parental combinations, or both can increase the efficiency of inducing cybrids in Citrus by electrofusion.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

scholarly journals The Association of an SNP in the EXOC4 Gene and Reproductive Traits Suggests Its Use as a Breeding Marker in Pigs

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 521
Author(s):  
Yingting He ◽  
Xiaofeng Zhou ◽  
Rongrong Zheng ◽  
Yao Jiang ◽  
Zhixiang Yao ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fragment Length Polymorphism ◽  
Regulatory Element ◽  
Reproductive Traits ◽  
Specific Sequence ◽  
Litter Weight ◽  
Potential Binding ◽  
Pcr Rflp ◽  
Teat Number ◽  
Myeloid Zinc Finger

In mammals, the exocyst complex component 4 (EXOC4) gene has often been reported to be involved in vesicle transport. The SNP rs81471943 (C/T) is located in the intron of porcine EXOC4, while six quantitative trait loci (QTL) within 5–10 Mb around EXOC4 are associated with ovary weight, teat number, total offspring born alive, and corpus luteum number. However, the molecular mechanisms between EXOC4 and the reproductive performance of pigs remains to be elucidated. In this study, rs81471943 was genotyped from a total of 994 Duroc sows, and the genotype and allele frequency of SNP rs81471943 (C/T) were statistically analyzed. Then, the associations between SNP rs81471943 and four reproductive traits, including number of piglets born alive (NBA), litter weight at birth (LWB), number of piglets weaned (NW), and litter weight at weaning (LWW), were determined. Sanger sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) were utilized to identify the rs81471943 genotype. We found that the genotype frequency of CC was significantly higher than that of CT and TT, and CC was the most frequent genotype for NBA, LWB, NW, and LWW. Moreover, 5′-deletion and luciferase assays identified a positive transcription regulatory element in the EXOC4 promoter. After exploring the EXOC4 promoter, SNP −1781G/A linked with SNP rs81471943 (C/T) were identified by analysis of the transcription activity of the haplotypes, and SNP −1781 G/A may influence the potential binding of P53, E26 transformation specific sequence -like 1 transcription factor (ELK1), and myeloid zinc finger 1 (MZF1). These findings provide useful information for identifying a molecular marker of EXOC4-assisted selection in pig breeding.

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes

1990 ◽  
Vol 10 (11) ◽  
pp. 5849-5856
Author(s):  
S E Sweigert ◽  
D Carroll
Keyword(s):  
Homologous Recombination ◽  
Xenopus Laevis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Coli Strain ◽  
Xenopus Laevis Oocytes ◽  
X Rays ◽  
Strand Breaks ◽  
X Ray ◽  
Dna Strand

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii

1983 ◽  
Vol 3 (6) ◽  
pp. 1070-1076
Author(s):  
S M Landfear ◽  
D McMahon-Pratt ◽  
D F Wirth
Keyword(s):  
Tandem Repeat ◽  
Blot Hybridization ◽  
Protozoan Parasite ◽  
Southern Blot Hybridization ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Southern Blot Hybridization Analysis ◽  
Leishmania Enriettii

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

Presence of Human Herpesvirus 8 DNA Sequences in Renal Transplantation-Associated Pleural Kaposi Sarcoma

1999 ◽  
Vol 123 (12) ◽  
pp. 1269-1273
Author(s):  
J. Javier Gómez-Román ◽  
J. Gonzalo Ocejo-Vinyals ◽  
Pablo Sánchez-Velasco ◽  
Francisco Leyva-Cobián ◽  
J. Fernando Val-Bernal
Keyword(s):  
Kidney Transplantation ◽  
Pleural Effusion ◽  
Dna Sequences ◽  
Blot Hybridization ◽  
Human Herpesvirus ◽  
Kaposi Sarcoma ◽  
Southern Blot Hybridization ◽  
Pulmonary Involvement ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8

Abstract Objective.—To describe one case of symptomatic skin and pleural Kaposi sarcoma (KS) associated with kidney transplantation. Diagnosis was supported by morphologic study and human herpesvirus 8 (HHV-8) detection in both tissues. Pulmonary involvement was not present. Design.—The presence of HHV-8 DNA sequences was proved using polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization. Setting.—Human herpesvirus 8 is found in most KS from patients with and without the acquired immunodeficiency syndrome. Clinically significant pulmonary infiltration by KS is diagnosed uncommonly antemortem, and pleural disease is exceptional. Patient.—A 49-year-old man who had renal transplant with immunosuppressive therapy (tacrolimus and prednisone) and developed a cutaneous KS. A pleural effusion appeared without pulmonary involvement. Both lesions disappeared when immunosuppressive drugs were suspended. Later, the pleural effusion and the cutaneous lesions reappeared. Pleural biopsy specimens showed KS infiltration. Outcome.—The patient refused treatment and was lost to follow-up. Results.—The skin and pleural biopsies showed a proliferation of spindle-shaped cells positive for CD34. The HHV-8 sequences were detected by nested PCR. No amplification was detected in uninvolved skin from the patient or in peripheral blood mononuclear cells from 10 healthy individuals used as controls. The Southern blot hybridization confirmed these results. Conclusions.—To our knowledge, this is the first report of HHV-8 in symptomatic pleural KS, which was probably associated with immunosuppression after kidney transplantation. The demonstration of HHV-8 DNA in biopsy material in the appropriate cells could be diagnostic when the morphologic setting is consistent with KS.

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