scholarly journals Centromere mitotic recombination in mammalian cells

2008 ◽  
Vol 181 (6) ◽  
pp. 885-892 ◽  
Author(s):  
Isabel Jaco ◽  
Andrés Canela ◽  
Elsa Vera ◽  
Maria A. Blasco
Keyword(s):  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Mitotic Recombination ◽  
Dna Methyltransferases ◽  
Centromeric Heterochromatin ◽  
Hybridization Technique ◽  
Centromeric Function ◽  
Epigenetic State ◽  
Chromosome Arm

Centromeres are special structures of eukaryotic chromosomes that hold sister chromatid together and ensure proper chromosome segregation during cell division. Centromeres consist of repeated sequences, which have hindered the study of centromere mitotic recombination and its consequences for centromeric function. We use a chromosome orientation fluorescence in situ hybridization technique to visualize and quantify recombination events at mouse centromeres. We show that centromere mitotic recombination occurs in normal cells to a higher frequency than telomere recombination and to a much higher frequency than chromosome-arm recombination. Furthermore, we show that centromere mitotic recombination is increased in cells lacking the Dnmt3a and Dnmt3b DNA methyltransferases, suggesting that the epigenetic state of centromeric heterochromatin controls recombination events at these regions. Increased centromere recombination in Dnmt3a,3b-deficient cells is accompanied by changes in the length of centromere repeats, suggesting that prevention of illicit centromere recombination is important to maintain centromere integrity in the mouse.

scholarly journals Identification of overlapping DNA-binding and centromere-targeting domains in the human kinetochore protein CENP-C.

1996 ◽  
Vol 16 (7) ◽  
pp. 3576-3586 ◽  
Author(s):  
C H Yang ◽  
J Tomkiel ◽  
H Saitoh ◽  
D H Johnson ◽  
W C Earnshaw
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
In Vitro Assays ◽  
Centromeric Heterochromatin ◽  
Structural Protein ◽  
Chromosomal Site

The kinetochore in eukaryotes serves as the chromosomal site of attachment for microtubules of the mitotic spindle and directs the movements necessary for proper chromosome segregation. In mammalian cells, the kinetochore is a highly differentiated trilaminar structure situated at the surface of the centromeric heterochromatin. CENP-C is a basic, DNA-binding protein that localizes to the inner kinetochore plate, the region that abuts the heterochromatin. Microinjection experiments using antibodies specific for CENP-C have demonstrated that this protein is required for the assembly and/or stability of the kinetochore as well as for a timely transition through mitosis. From these observations, it has been suggested that CENP-C is a structural protein that is involved in the organization or the kinetochore. In this report, we wished to identify and map the functional domains of CENP-C. Analysis of CENP-C truncation mutants expressed in vivo demonstrated that CENP-C possesses an autonomous centromere-targeting domain situated at the central region of the CENP-C polypeptide. Similarly, in vitro assays revealed that a region of CENP-C with the ability to bind DNA is also located at the center of the CENP-C molecule, where it overlaps the centromere-targeting domain.

scholarly journals Wheat, Rye, and Barley Genomes Can Associate during Meiosis in Newly Synthesized Trigeneric Hybrids

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 113
Author(s):  
María-Dolores Rey ◽  
Carmen Ramírez ◽  
Azahara C. Martín
Keyword(s):  
Chromosome Segregation ◽  
Genome Duplication ◽  
Triticum Turgidum ◽  
Aegilops Tauschii ◽  
Genomic In Situ Hybridization ◽  
Hordeum Chilense ◽  
Genomic Constitution ◽  
Chromosome Associations ◽  
High Level

Polyploidization, or whole genome duplication (WGD), has an important role in evolution and speciation. One of the biggest challenges faced by a new polyploid is meiosis, in particular, discriminating between multiple related chromosomes so that only homologs recombine to ensure regular chromosome segregation and fertility. Here, we report the production of two new hybrids formed by the genomes of species from three different genera: a hybrid between Aegilops tauschii (DD), Hordeum chilense (HchHch), and Secale cereale (RR) with the haploid genomic constitution HchDR (n = 7× = 21); and a hybrid between Triticum turgidum spp. durum (AABB), H. chilense, and S. cereale with the constitution ABHchR (n = 7× = 28). We used genomic in situ hybridization and immunolocalization of key meiotic proteins to establish the chromosome composition of the new hybrids and to study their meiotic behavior. Interestingly, there were multiple chromosome associations at metaphase I in both hybrids. A high level of crossover (CO) formation was observed in HchDR, which shows the possibility of meiotic recombination between the different genomes. We succeeded in the duplication of the ABHchR genome, and several amphiploids, AABBHchHchRR, were obtained and characterized. These results indicate that recombination between the genera of three economically important crops is possible.

scholarly journals Aneuploid abortion correlates positively with MAD1 overexpression and miR-125b down-regulation

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Juan Zhao ◽  
Hui Li ◽  
Guangxin Chen ◽  
Lijun Du ◽  
Peiyan Xu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Checkpoint ◽  
Early Embryo ◽  
Vital Role ◽  
Control Group ◽  
Embryo Abortion ◽  
Protein Levels ◽  
Mitotic Checkpoint Complex

Abstract Background Aneuploidy is the most frequent cause of early-embryo abortion. Any defect in chromosome segregation would fail to satisfy the spindle assembly checkpoint (SAC) during mitosis, halting metaphase and causing aneuploidy. The mitotic checkpoint complex (MCC), comprising MAD1, MAD2, Cdc20, BUBR1 and BUB3, plays a vital role in SAC activation. Studies have confirmed that overexpression of MAD2 and BUBR1 can facilitate correct chromosome segregation and embryo stability. Research also proves that miR-125b negatively regulates MAD1 expression by binding to its 3′UTR. However, miR-125b, Mad1 and Bub3 gene expression in aneuploid embryos of spontaneous abortion has not been reported to date. Methods In this study, embryonic villi from miscarried pregnancies were collected and divided into two groups (aneuploidy and euploidy) based on High-throughput ligation-dependent probe amplification (HLPA) and Fluorescence in situ hybridization (FISH) analyses. RNA levels of miR-125b, MAD1 and BUB3 were detected by Quantitative real-time PCR (qRT-PCR); protein levels of MAD1 and BUB3 were analysed by Western blotting. Results statistical analysis (p < 0.05) showed that miR-125b and BUB3 were significantly down-regulated in the aneuploidy group compared to the control group and that MAD1 was significantly up-regulated. Additionally, the MAD1 protein level was significantly higher in aneuploidy abortion villus, but BUB3 protein was only mildly increased. Correlation analysis revealed that expression of MAD1 correlated negatively with miR-125b. Conclusion These results suggest that aneuploid abortion correlates positively with MAD1 overexpression, which might be caused by insufficient levels of miR-125b. Taken together, our findings first confirmed the negative regulatory mode between MAD1 and miR-125b, providing a basis for further mechanism researches in aneuploid abortion.

scholarly journals Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrio parahaemolyticus in Oysters and Mussels

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1502
Author(s):  
Jorge García-Hernández ◽  
Manuel Hernández ◽  
Yolanda Moreno
Keyword(s):  
In Situ Hybridization ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent In Situ Hybridization ◽  
Potential Method ◽  
Viable Count ◽  
Hybridization Technique ◽  
Fish Technique ◽  
Viable Cells

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication

1995 ◽  
Vol 108 (3) ◽  
pp. 927-934 ◽  
Author(s):  
M. Starborg ◽  
E. Brundell ◽  
K. Gell ◽  
C. Larsson ◽  
I. White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Mitotic Cell ◽  
Yeast Cells ◽  
Metaphase Chromosomes ◽  
Licensing Factor ◽  
Mammalian Homologue ◽  
Initiation Of Dna Replication

We have analyzed the expression of the murine P1 gene, the mammalian homologue of the yeast MCM3 protein, during the mitotic cell cycle. The MCM3 protein has previously been shown to be of importance for initiation of DNA replication in Saccharomyces cerevisiae. We found that the murine P1 protein was present in the nuclei of mammalian cells throughout interphase of the cell cycle. This is in contrast to the MCM3 protein, which is located in the nuclei of yeast cells only between the M and the S phase of the cell cycle. Detailed analysis of the intranuclear localization of the P1 protein during the cell cycle revealed that it accumulates transiently in the heterochromatic regions towards the end of G1. The accumulation of the P1 protein in the heterochromatic regions prior to activation of DNA replication suggests that the mammalian P1 protein is also of importance for initiation of DNA replication. The MCM2-3.5 proteins have been suggested to represent yeast equivalents of a hypothetical replication licensing factor initially described in Xenopus. Our data support this model and indicate that the murine P1 protein could function as replication licensing factor. The chromosomal localization of the P1 gene was determined by fluorescence in situ hybridization to region 6p12 in human metaphase chromosomes.

Microbial ecology of sulfate-reducing bacteria in wastewater biofilms analyzed by microelectrodes and fish (fluorescent in situ hybridization) technique

1999 ◽  
Vol 39 (7) ◽  
pp. 41-47 ◽  
Author(s):  
Satoshi Okabe ◽  
Hisashi Satoh ◽  
Tsukasa Itoh ◽  
Yoshimasa Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Sulfate Reduction ◽  
Sulfate Reducing Bacteria ◽  
Hybridization Technique ◽  
Concentration Profiles ◽  
Reduction Zone ◽  
Sulfate Reducing ◽  
Reducing Bacteria ◽  
Culture Dependent

The vertical distribution of sulfate-reducing bacteria (SRB) in microaerophilic wastewater biofilms grown on fully submerged rotating disk reactors (RDR) was determined by the conventional culture-dependent MPN method and in situ hybridization of fluorescently-labelled 16S rRNA-targeted oligonucleotide probes for SRB in parallel. Chemical concentration profiles within the biofilm were also measured using microelectrodes for O2, S2-, NO3- and pH. In situ hybridization revealed that the SRB probe-stained cells were distributed throughout the biofilm even in the oxic surface zone in all states from single scattered cells to clustered cells. The higher fluorescence intensity and abundance of SRB probe-stained cells were found in the middle part of the biofilm. This result corresponded well with O2 and H2S concentration profiles measured by microelectrodes, showing sulfate reduction was restricted to a narrow anaerobic zone located about 500 μm below the biofilm surface. Results of the MPN and potential sulfate reducing activity (culture-dependent approaches) indicated a similar distribution of cultivable SRB in the biofilm. The majority of the general SRB probe-stained cells were hybridized with SRB 660 probe, suggesting that one important member of the SRB in the wastewater biofilm could be the genus Desulfobulbus. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from O2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium.

Using genomic slot blot hybridization to assess intergeneric Saccharum x Erianthus hybrids (Andropogoneae – Saccharinae)

Genome ◽  
1997 ◽  
Vol 40 (4) ◽  
pp. 428-432 ◽  
Author(s):  
P. Besse ◽  
C. L. McIntyre ◽  
D. M. Burner ◽  
C. G. de Almeida
Keyword(s):  
Genomic Dna ◽  
Blot Hybridization ◽  
Female Parent ◽  
Saccharum Officinarum ◽  
Rapid Screening ◽  
Hybridization Technique ◽  
Slot Blot ◽  
Saccharum Species ◽  
Slot Blot Hybridization

The use of genomic slot blot hybridization enabled the differentiation of hybrids from selfs in Saccharum × Erianthus intergeneric crosses in which Saccharum was used as the female parent. Based on the genomic in situ hybridization technique, slot blots of DNA from the parents and the progeny were blocked with the Saccharum parent DNA and hybridized with the labelled male Erianthus genomic DNA. This technique allowed a rapid screening for hybrids and was sensitive enough to detect a 1/20 dilution of Erianthus in Saccharum DNA, which should enable the detection of most partial hybrids. The genomic slot blot hybridization technique was shown to be potentially useful for assessing crosses involving Saccharum species with either Old World Erianthus section Ripidium or North American Erianthus (= Saccharum) species. The effectiveness of the technique was assessed on 144 progeny of a Saccharum officinarum × Erianthus arundinaceus cross, revealing that 43% of the progeny were selfs. The importance of this test as a tool to support intergeneric breeding programs is discussed.Key words: slot blot, Erianthus, genomic DNA, Saccharum, sugarcane.

scholarly journals Monosomy 12 and deletion of 13q34 in a case of chronic lymphocytic leukemia with concomitant lung cancer

2010 ◽  
Vol 67 (10) ◽  
pp. 864-866 ◽  
Author(s):  
Darko Antic ◽  
Marija Dencic-Fekete ◽  
Dragica Tomin ◽  
Irena Djunic
Keyword(s):  
Lung Cancer ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lung Carcinoma ◽  
Prognostic Significance ◽  
Specific Treatment ◽  
Lymphocytic Leukemia ◽  
Simultaneous Occurrence ◽  
Hybridization Technique

Background. We described a patient with chronic lymphocytic leukemia (CLL) and lung cancer and unusual chromosomal aberrations. Case report. At the same time with the diagnosis of B-cell CLL, squamocellular lung carcinoma diagnosis was established. Using interphase fluoresecence in situ hybridization technique (FISH) we detected monosomy 12 and deletion of 13q34 occured in the same clone. One month after the beginning of examination the patient died unexpectedly during sleep immediately before we applied a specific treatment for CLL or lung carcinoma. Conclusion. Simultaneous occurrence of monosomy 12 and deletion of 13q34 in a patient with B-cell CLL has been described only once before, but as a part of a complex karyotype. The prognostic significance of these abnormalities remains uncertain.

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