261 LABELING SEX-SPECIFIC DNA SEQUENCES IN MAMMALIAN SPERM

2008 ◽  
Vol 20 (1) ◽  
pp. 210
Author(s):  
B. A. Didion ◽  
R. Bleher
Keyword(s):  
Dna Sequences ◽  
Somatic Cells ◽  
Complete Separation ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Telomeric Sequences ◽  
Synthetic Dna ◽  
Y Chromosomes ◽  
Boar Sperm ◽  
The Difference

While flow cytometric separation of X- andY-chromosome- bearing sperm has advanced to the point of acceptance in the commercial production of sex-preselected cattle, it is important to continue researching this area to improve efficiencies. For example, the difference in DNA sequence between the X- andY-chromosomes has merit as a foundation for an alternative sperm sexing approach that could enable the complete separation and use of an entire ejaculate. We used synthetic DNA mimics conjugated to a fluorescent dye for in situ detection of Y-chromosomes in metaphase preparations of porcine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are synthetic compounds with higher affinity and stability than conventional DNA probes and are used as specific hybridization probes to complementary DNA. The application of PNA probes was demonstrated previously in telomere analysis studies, and we confirmed their efficacy using a CY3-(CCCTAA)3 PNA to probe bull and boar sperm telomeric sequences. Using male porcine somatic cells and theY-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13-15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation of metaphase chromosomes with the PNA produced a localized signal on theY-chromosome. No signals were present when chromosomes of porcine female somatic cells were incubated with the PNA probes. Because chromosomes occupy non-random territories in all cell nuclei including those in sperm, we expected to find centrally located signals in 50% of fixed boar sperm when these were treated with the same PNA as used for the somatic cells. We found the signals present in 161 of 302 (53.3%) sperm to consist of a single, centrally located, round fluorescent dot in the sperm head. Further research is required to establish the uptake of PNA in live sperm toward evaluation of this approach for sperm sexing.

263 LABELING BOVINE SPERM Y-CHROMOSOME SEQUENCE USING DNA MIMICS

2009 ◽  
Vol 21 (1) ◽  
pp. 229 ◽  
Author(s):  
B. A. Didion ◽  
R. Bleher
Keyword(s):  
Y Chromosome ◽  
Somatic Cells ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Synthetic Dna ◽  
Bovine Sperm ◽  
Hybridization Probes ◽  
Y Chromosomes ◽  
Chromosome Sequence ◽  
In Situ Detection

Flow cytometric separation of X- and Y-chromosome bearing bovine sperm is an accepted technology for use at the commercial level. Nevertheless it is important to continue researching the area of gender-preselected sperm for improved efficiencies. We used a synthetic DNA mimic conjugated to a fluorescent dye for in situ detection of Y chromosomes in metaphase preparations of bovine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are a type of DNA mimic having a higher affinity and stability than conventional DNA probes and are used as hybridization probes to complementary DNA. Using male bovine somatic cells and the Y-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13 to 15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation (~1 h) of metaphase chromosomes with the PNA produced a localized signal on the Y-chromosome. No signals were observed when chromosomes of female bovine somatic cells were incubated with the same PNA probe. Because chromosomes occupy non-random territories in all cell nuclei, including sperm, we proposed to find centrally-located signals in 50% of fixed bovine sperm when treated with the same PNA as used for the somatic cells. As expected, we found the PNA signals present in 50% sperm (23/43) existing as a single, centrally-located, round fluorescent dot in the sperm head. Validation studies were also conducted using bovine sperm previously flow sorted into X or Y populations, and we found the signals in accordance to an expected signal present using the PNA (146/165 or 88.5% with PNA signal in presorted Y sperm heads and 13/174 or 7.5% with PNA signal in presorted X sperm heads).

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.

scholarly journals Telomeric DNA sequences in beetle taxa vary with species richness

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniela Prušáková ◽  
Vratislav Peska ◽  
Stano Pekár ◽  
Michal Bubeník ◽  
Lukáš Čížek ◽  
...  
Keyword(s):  
Species Richness ◽  
Dna Sequences ◽  
Genome Instability ◽  
Telomeric Sequence ◽  
Dna Repeats ◽  
Telomeric Dna ◽  
Common Pattern ◽  
Telomeric Sequences ◽  
Genomic Changes ◽  
Protective Structures

AbstractTelomeres are protective structures at the ends of eukaryotic chromosomes, and disruption of their nucleoprotein composition usually results in genome instability and cell death. Telomeric DNA sequences have generally been found to be exceptionally conserved in evolution, and the most common pattern of telomeric sequences across eukaryotes is (TxAyGz)n maintained by telomerase. However, telomerase-added DNA repeats in some insect taxa frequently vary, show unusual features, and can even be absent. It has been speculated about factors that might allow frequent changes in telomere composition in Insecta. Coleoptera (beetles) is the largest of all insect orders and based on previously available data, it seemed that the telomeric sequence of beetles varies to a great extent. We performed an extensive mapping of the (TTAGG)n sequence, the ancestral telomeric sequence in Insects, across the main branches of Coleoptera. Our study indicates that the (TTAGG)n sequence has been repeatedly or completely lost in more than half of the tested beetle superfamilies. Although the exact telomeric motif in most of the (TTAGG)n-negative beetles is unknown, we found that the (TTAGG)n sequence has been replaced by two alternative telomeric motifs, the (TCAGG)n and (TTAGGG)n, in at least three superfamilies of Coleoptera. The diversity of the telomeric motifs was positively related to the species richness of taxa, regardless of the age of the taxa. The presence/absence of the (TTAGG)n sequence highly varied within the Curculionoidea, Chrysomeloidea, and Staphylinoidea, which are the three most diverse superfamilies within Metazoa. Our data supports the hypothesis that telomere dysfunctions can initiate rapid genomic changes that lead to reproductive isolation and speciation.

Accuracy and precision in stock separation of north-east Arctic and Norwegian coastal cod by otoliths - comparing readings, image analyses and a genetic method

2005 ◽  
Vol 56 (5) ◽  
pp. 753 ◽  
Author(s):  
Erik Berg ◽  
Tuula H. Sarvas ◽  
Alf Harbitz ◽  
Svein Erik Fevolden ◽  
Arnt Børre Salberg
Keyword(s):  
Visual Inspection ◽  
Gadus Morhua ◽  
Atlantic Cod ◽  
Complete Separation ◽  
Arctic Cod ◽  
Genetic Method ◽  
North East ◽  
Accuracy And Precision ◽  
Growth Zones ◽  
The Difference

The distinction between north-east Arctic cod and Norwegian coastal cod, two major groups of Atlantic cod (Gadus morhua L.), has for many years been based on different distance and shape similarities between the two first translucent growth zones in the otoliths, subjectively decided by visual inspection in a binocular. To analyse the certainty of this technique, four independent readers have classified 263 cod otoliths in total from five different geographical areas. For three of the readers, between 82% and 89% of the classification results coincided with independent results based on genetic analyses. Further, 38 cod otoliths, where the readers were certain of the classification (21 north-east Arctic cod and 17 coastal cod) were classified by several image analysis methods. A complete separation was obtained by using the ratio of the circumferences of the two zones, providing a typical ratio of approximately 2 for coastal and 1.5 for north-east Arctic cod. The otolith method for separating the two types of cod has been considered adequately accurate in assessing the two stocks of cod. However, the method is sensitive to subjective interpretation, and action needs to be taken to minimise the difference in interpretation among otolith readers.

Insertion and excision of Caenorhabditis elegans transposable element Tc1

1988 ◽  
Vol 8 (2) ◽  
pp. 737-746
Author(s):  
D Eide ◽  
P Anderson
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Transposable Element ◽  
Dna Sequences ◽  
Germ Line ◽  
Consensus Sequence ◽  
Somatic Cells ◽  
Chain Gene ◽  
Imprecise Excision ◽  
Insertion Sites

The transposable element Tc1 is responsible for most spontaneous mutations that occur in Caenorhabditis elegans variety Bergerac. We investigated the genetic and molecular properties of Tc1 transposition and excision. We show that Tc1 insertion into the unc-54 myosin heavy-chain gene was strongly site specific. The DNA sequences of independent Tc1 insertion sites were similar to each other, and we present a consensus sequence for Tc1 insertion that describes these similarities. We show that Tc1 excision was usually imprecise. Tc1 excision was imprecise in both germ line and somatic cells. Imprecise excision generated novel unc-54 alleles that had amino acid substitutions, amino acid insertions, and, in certain cases, probably altered mRNA splicing. The DNA sequences remaining after Tc1 somatic excision were the same as those remaining after germ line excision, but the frequency of somatic excision was at least 1,000-fold higher than that of germ line excision. The genetic properties of Tc1 excision, combined with the DNA sequences of the resulting unc-54 alleles, demonstrated that excision was dependent on Tc1 transposition functions in both germ line and somatic cells. Somatic excision was not regulated in the same strain-specific manner as germ-line excision was. In a genetic background where Tc1 transposition and excision in the germ line was not detectable, Tc1 excision in the soma still occurred at high frequency.

The Synthesis and Migration of Nuclear Proteins during Mitosis and differentiation of Cells in Rats

1965 ◽  
Vol s3-106 (75) ◽  
pp. 229-240
Author(s):  
R. T. SIMS
Keyword(s):  
Granular Cell ◽  
Nuclear Proteins ◽  
Photographic Emulsion ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei ◽  
Cell Layers ◽  
The Difference ◽  
Mitotic Figures ◽  
And Migration ◽  
Silver Grains

Hooded rats were given an intraperitoneal injection of 3H-tyrosine, and killed in pairs 10 min, 30 min, 12 h, 36 h, 7 days, and 30 days later. A piece of skin with white growing hair, and the tongue, were taken from each animal and radioautographs were prepared. Silver grains were counted over whole nuclei and whole mitotic figures of the germinal cells and whole nuclei of differentiating cells of both tissues. It was found that the interphase nuclei have significantly more silver grains over them than the chromosomes at all stages of mitosis and there are virtually no grains over metaphase, anaphase, and early telophase chromosomes in both tissues of all the animals killed up to 36 h after the injection. The difference between the grain counts over the interphase nuclei and the chromosomes of dividing cells is at least 20-fold at 30 min in the hair matrix, at least 5-fold at 30 min in the tongue and at 36 h in both tissues. It was established that the differences observed between the radioactivities of the nuclei and chromosomes of mitotic figures are real from estimates of: the radioactivity of the cell cytoplasm, volumes of the metaphase chromosomes and interphase nuclei within 1µ of the photographic emulsion, and the volumes of cytoplasm separating the photographic emulsion and these structures. No protein synthesis was demonstrable in the chromosomes during metaphase, anaphase, and early telophase. Nuclear proteins leave the chromosomes during prophase and prometaphase and return to the nucleus during late telophase. The cells in the matrix and upper bulb of the growing hair follicle and those in the germinal, prickle, and granular cell layers of the tongue are in different functional states; 30 min after injection of 3H-tyrosine they have different amounts of it in their nuclear proteins. It is suggested that the amount incorporated into each nucleus is related to the rate at which proteins are being synthesized by the cell.

In the Aftermath of the “Myriad Case” – Myriad Is Denied Preliminary Injunction Against Ambry Genetics

2014 ◽  
Vol 5 (3) ◽  
pp. 407-412
Author(s):  
Emanuela Gambini
Keyword(s):  
Dna Sequences ◽  
Case Note ◽  
District Court ◽  
Central Division ◽  
University Of Utah ◽  
Synthetic Dna ◽  
Preliminary Injunction ◽  
The U.S

On March 10, 2014, the U.S. District Court of Utah, Central Division, decided in University of Utah Research Foundation, et al., v. Ambry Genetics Corporation, holding that “Plaintiffs are not entitled to a preliminary injunction”, as they “are unable to establish that they are likely to succeed on the merits of the claims” nor “that the equitable factors support issuance of the requested injunction”.This case note gives an overview of the U.S. District Court's of Utah memorandum decision and order denying plaintiffs’ motion for preliminary injunction and discusses its implications for the implementation of the criterion of isolation to “synthetic” DNA sequences, such as primers and probes.

scholarly journals The LMO2 oncogene regulates DNA replication in hematopoietic cells

2016 ◽  
Vol 113 (5) ◽  
pp. 1393-1398 ◽  
Author(s):  
Marie-Claude Sincennes ◽  
Magali Humbert ◽  
Benoît Grondin ◽  
Véronique Lisi ◽  
Diogo F. T. Veiga ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Sequences ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Dna Polymerase Delta ◽  
Thymocyte Development ◽  
Erythroid Progenitors ◽  
Acute Leukemias ◽  
Synthetic Dna

Oncogenic transcription factors are commonly activated in acute leukemias and subvert normal gene expression networks to reprogram hematopoietic progenitors into preleukemic stem cells, as exemplified by LIM-only 2 (LMO2) in T-cell acute lymphoblastic leukemia (T-ALL). Whether or not these oncoproteins interfere with other DNA-dependent processes is largely unexplored. Here, we show that LMO2 is recruited to DNA replication origins by interaction with three essential replication enzymes: DNA polymerase delta (POLD1), DNA primase (PRIM1), and minichromosome 6 (MCM6). Furthermore, tethering LMO2 to synthetic DNA sequences is sufficient to transform these sequences into origins of replication. We next addressed the importance of LMO2 in erythroid and thymocyte development, two lineages in which cell cycle and differentiation are tightly coordinated. Lowering LMO2 levels in erythroid progenitors delays G1-S progression and arrests erythropoietin-dependent cell growth while favoring terminal differentiation. Conversely, ectopic expression in thymocytes induces DNA replication and drives these cells into cell cycle, causing differentiation blockade. Our results define a novel role for LMO2 in directly promoting DNA synthesis and G1-S progression.

scholarly journals Repetitive DNA sequences accelerate molecular cytogenetic research in plants with small chromosomes

2019 ◽  
Vol 24 (2) ◽  
pp. 82
Author(s):  
Agus Budi Setiawan ◽  
Ari Wibowo ◽  
Chee How Teo ◽  
Shinji Kikuchi ◽  
Takato Koba
Keyword(s):  
Repetitive Dna ◽  
Dna Sequences ◽  
5S Rdna ◽  
Repetitive Dna Sequences ◽  
Type I ◽  
Chromosome Identification ◽  
Metaphase Chromosomes ◽  
Homologous Chromosomes ◽  
Centromeric Sequence ◽  
Subtelomeric Sequence

Repetitive DNA sequences are highly abundant in plant genomes and are favorable probes for chromosome identification in plants. However, it is difficult to conduct studies on the details of metaphase chromosome structures in plants with small chromosomes due to their highly condensed status. Therefore, identification of homologous chromosomes for karyotyping and analyzing chromosome structures is a challenging issue for cytogeneticists without specific probes and precise chromosome stages. In this study, five repetitive DNA probes, i.e., 5S and 45S ribosomal DNAs (rDNAs), melon centromeric sequence (Cmcent), cucumber subtelomeric sequence (Type I), and microsatellite (CT)10 repeats, were used to identify primary constrictions and homologous chromosomes for karyotyping. Four and two loci of 45S rDNA were respectively observed on metaphase and pachytene chromosomes of Abelia × grandiflora. Cmcent was detected on both primary constrictions of melon pachytene and metaphase chromosomes. Furthermore, one pair of 5S rDNA signals were hybridized on melon metaphase chromosomes. Eight and two loci of 45S and 5S rDNA were respectively detected on cucumber chromosomes. Type I and (CT)10 probes were specifically hybridized on subtelomeric and interstitial regions on the chromosomes, respectively. These results suggest that repetitive DNA sequences are versatile probes for chromosome identification in plants with small chromosomes, particularly for karyotyping analyses.

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