scholarly journals Nick-translation of metaphase chromosomes: in vitro labeling of nuclease-hypersensitive regions in chromosomes.

1985 ◽  
Vol 82 (3) ◽  
pp. 854-858 ◽  
Author(s):  
M. T. Kuo ◽  
W. Plunkett
Keyword(s):  
Nick Translation ◽  
Metaphase Chromosomes

Some Structural Features of Metaphase Chromosomes of Mice and Men

1967 ◽  
Vol 25 ◽  
pp. 190-191
Author(s):  
Godfrey C. Hoskins ◽  
Betty B. Hoskins
Keyword(s):  
Electron Microscopy ◽  
Electron Micrographs ◽  
Structural Features ◽  
Metaphase Chromosomes ◽  
The Future ◽  
Of Mice And Men ◽  
Mouse Cells ◽  
Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

DNA sequence mapping in interphase and metaphase chromosomes by fluorescence in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 496-497
Author(s):  
Barbara Trask ◽  
Susan Allen ◽  
Anne Bergmann ◽  
Mari Christensen ◽  
Anne Fertitta ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Dual Band ◽  
Nick Translation ◽  
Metaphase Chromosomes ◽  
Band Pass ◽  
Texas Red ◽  
Fluorescent Spot

Using fluorescence in situ hybridization (FISH), the positions of DNA sequences can be discretely marked with a fluorescent spot. The efficiency of marking DNA sequences of the size cloned in cosmids is 90-95%, and the fluorescent spots produced after FISH are ≈0.3 μm in diameter. Sites of two sequences can be distinguished using two-color FISH. Different reporter molecules, such as biotin or digoxigenin, are incorporated into DNA sequence probes by nick translation. These reporter molecules are labeled after hybridization with different fluorochromes, e.g., FITC and Texas Red. The development of dual band pass filters (Chromatechnology) allows these fluorochromes to be photographed simultaneously without registration shift.

51 DEVELOPMENT OF EQUINE CLONED EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES RECEIVING ADULT DERMAL FIBROBLAST CELL NUCLEI

2009 ◽  
Vol 21 (1) ◽  
pp. 125
Author(s):  
M. Skrzyszowska ◽  
M. Samiec ◽  
W. Mlodawska ◽  
J. Kochan ◽  
A. Okolski ◽  
...  
Keyword(s):  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Vero Cells ◽  
Contact Inhibition ◽  
Fibroblast Cells ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Condensed Chromosome ◽  
Donor Cells

The purpose of our study was to determine the in vitro developmental competences of equine NT embryos reconstructed with adult dermal fibroblast cells. Frozen/thawed fibroblast cells, whose mitotic cycle had been synchronized at G1/G0 stages through a contact inhibition of their migration and proliferative activity under total confluency, were used as a source of nuclear donor cells in the somatic cell cloning procedure. In vitro-matured oocytes were used as recipient cells for fibroblast cell nuclei. The compact cumulus–oocyte complexes (cpCOCs) were collected from abattoir-derived mare ovaries and selected for in vitro maturation. The cpCOCs were cultured in TC-199 medium supplemented with 5 mU mL–1 follicle-stimulating hormone (FSH), 10% fetal bovine serum (FBS) and 75 μg mL–1 kanamycin monosulfate (kanamycin A) for 30 h at 38.2°C in a 100% water-saturated atmosphere of 5% CO2 and 95% air. Cumulus-denuded in vitro-matured oocytes were incubated in the maturation medium supplemented with 0.4 μg mL–1 demecolcine for 40 min. The treated oocytes were subsequently transferred into TC-199 medium containing 4 mg mL–1 BSA-V and 5 μg mL–1 cytochalasin B. Metaphase chromosomes, which had been allocated into the chemically-induced protrusion of the plasma membrane, were removed microsurgically. The chemically-assisted enucleation was accomplished by gently aspirating the ooplasmic cone, which contained the condensed chromosome mass, with the aid of a beveled micropipette. The single nuclear donor cells were inserted into perivitelline space of previously enucleated oocytes. Fibroblast cell-ooplast couplets were fused with two consecutive DC pulses of 2.4 kV cm–1 for 30 μs. After a 1.5-h delay, nuclear transfer-derived oocytes were chemically activated by exposure to 5 μm L–1 calcium ionomycin for 5 to 7 min, followed by their incubation in B2 medium with addition of 2 mm L–1 6-dimethylaminopurine (6-DMAP) for 4 h. Reconstructed embryos were in vitro cultured in B2 medium for 2 days. Afterwards, cleaved embryos were co-cultured with Vero cells in B2 medium supplemented with 10% FBS for 5 to 6 days up to morula/blastocyst stages. From among 88 in vitro cultured cpCOCs, 55 (62.5%) acquired meiotic nuclear and cytoplasmic maturity state after reaching the Metaphase II stage. A total of 55 enucleated oocytes underwent reconstruction and 44/55 (80.0%) were successfully fused with nuclear donor cells. Out of 44 cultured NT embryos, 21 (47.7%) were cleaved. The frequencies of cloned embryos that reached the morula and blastocyst stages were 6/44 (13.6%) and 3/44 (6.8%), respectively. In conclusion, the cell nuclei of in vitro cultured adult dermal fibroblast cells, which had undergone the contact inhibition, were able to direct the preimplantation development of equine cloned embryos to morula and blastocyst stages. This work was supported by the Scientific Net of Animal Reproduction Biotechnology.

INFLUENCE OF TEMPERATURE ON THE INDUCTION AND REPAIR OF RADIATION INDUCED ABERRATIONS IN THE HUMAN CHROMOSOME

1971 ◽  
Vol 13 (2) ◽  
pp. 364-368 ◽  
Author(s):  
K. C. Bora ◽  
L. Soper
Keyword(s):  
Human Chromosome ◽  
Treatment Time ◽  
Human Peripheral Blood ◽  
Metaphase Chromosomes ◽  
Similar Treatment ◽  
Aberration Frequency ◽  
Enhanced Sensitivity ◽  
Influence Of Temperature On ◽  
Radiation Induced

Investigations were carried out by (i) X-irradiating human peripheral blood lymphocyte cultures in vitro at 37 or 5 °C with 306 R at 102 R/min, and maintaining these at the respective temperatures for 65 min following irradiation, and before incubation at 37 °C, and by (ii) reversing the temperature of half the cultures immediately or 30 min following irradiation. Metaphase chromosomes were analysed in 54-hr cultures for induced aberrations. Aberration frequency was significantly higher in cultures irradiated at 37 °C and maintained at 37 °C for 65 min following irradiation than in cultures receiving parallel treatments at 5 °C. Reversal of temperature from 37 to 5°C and vice versa resulted in intermediate frequencies. Reduction of postirradiation treatment time from 65 to 30 min at 37 °C before reversal to 5 °C did not influence the exchange frequency. However, similar treatment at 5 °C and reversal to 37° showed an increase. It is suggested that the increase in the aberration frequency at high temperature is not due to an enhanced sensitivity of the human chromosome to radiation, but to an influence on the repair and exchange formation processes.

INSECT CHROMOSOME ISOLATION: A METHOD FOR METAPHASE CHROMOSOME HARVEST FROM CULTURED CELLS OF THE MOSQUITO AEDES ALBOPICTUS

1981 ◽  
Vol 23 (3) ◽  
pp. 453-457 ◽  
Author(s):  
Asit B. Mukherjee ◽  
Lorraine DeGiorgio
Keyword(s):  
Cell Line ◽  
Aedes Albopictus ◽  
Metaphase Chromosome ◽  
Cultured Cells ◽  
Modified Technique ◽  
Metaphase Chromosomes ◽  
Chromosome Isolation

A modified technique for bulk isolation of metaphase chromosomes from an in vitro cell line of the mosquito Aedes albopictus (Skuse) is described.

scholarly journals Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse.

1986 ◽  
Vol 102 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
H J Clarke ◽  
Y Masui
Keyword(s):  
Morphological Changes ◽  
Small Mass ◽  
Sperm Nucleus ◽  
Metaphase Chromosomes ◽  
Oocyte Meiosis ◽  
Maturation In Vitro ◽  
Sperm Penetration ◽  
Sperm Nuclei ◽  
Metaphase I

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.

scholarly journals Clonal Propagation and Karyomorphological Study of Solanum torvum Swartz: An Ethnobotanical Species of Tripura, India

2018 ◽  
Vol 28 (1) ◽  
pp. 69-76
Author(s):  
H Reshmi Singha ◽  
Sangram Sinha ◽  
Rabindra Kumar Sinha
Keyword(s):  
Clonal Propagation ◽  
Culture Media ◽  
Shoot Proliferation ◽  
Induction Phase ◽  
Root Induction ◽  
Metaphase Chromosomes ◽  
Solanum Torvum ◽  
Regenerated Plants ◽  
Bud Induction

An efficient method of clonal propagation through nodal culture of Solanum torvum Swartz. is described. Different concentrations of BAP/Kn alone or in combination with IAA were tested for direct shoot bud induction and proliferation. Lower concentration of BAP/Kn alone produced better shoot proliferation and elongation. Maximum number of shoot proliferation was achieved from MS supplemented with Kn 0.5 mg/l with an average 4.0 ± 1.41 shoots during 28 days of culture. Addition of IAA to the culture media in combination with BAP/ Kn significantly reduced the number of shoot formation. Regenerated plants also produced roots during subsequent culture in the same media supplemented with BAP/Kn alone or in combination with IAA. The easy nature of in vitro rooting of S. torvum was recorded without any separate root induction phase. Regenerated plants were successfully transferred to the field condition. Clonal feature was cytologically confirmed through the study of mitotic metaphase chromosomes of regenerated plants which reveals 2n = 24 somatic chromosomes. Comparative karyomorphological details between the mother and regenerated plants of S. torvum revealed close similarity in their chromosomal complements and falls under the category of "1B" Stebbin’s symmetric index suggesting true to type nature of the regenerated plant.Plant Tissue Cult. & Biotech. 28(1): 69-76, 2018 (June)

Sign in / Sign up

Export Citation Format

Share Document