Lilia López-Cánovas

Zinc-imidazole negative staining of chromosomal-sized DNA molecules in agarose minigels.

We standardized the zinc-imidazole negative staining method for detecting chromosomal-sized DNA molecules separated by pulsed field minigel electrophoresis. The best experimental conditions were as follows: separating large DNA molecules in minigels of 0.4 cm thickness, further incubating them with 40 mM ZnSO4 solution, and finally incubating them with 0.1 and 2 M imidazole solutions successively. The lowest yeast cells/miniplug useful in DNA band detection was 3 x 10(7) cells, as occurred with ethidium bromide-stained minigel. Electrophoresis patterns were visualized as colorless bands contrasting against a white background after illuminating the minigel with white light. This negative staining method is nontoxic and preserves the chemical integrity of the DNA molecules.

Adapting to contour-clamped homogeneous electric field minichamber technology the PulseNet protocols to resolve XbaI–DNA fragments of Salmonella serotype Braenderup

We propose cost-effective protocols for preparing and resolving the PulseNet universal DNA size standard in contour-clamped homogeneous electric field (CHEF) minichambers. Intact DNA molecules were prepared with protease-free solutions, and electrophoresis separations of the DNA standards needed 5.5h, giving band pattern resolutions similar to those attained with the PulseNet protocols standardized in CHEF chambers.

Comparative Study of Three Methods for Non-radioactive In Vivo DNA Labeling in Escherichia coli Using Nucleoside Analogs

Abstract In the present work, a comparative study of 5-FdUrd, thy -, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E. coli DH5α cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd. According to the colorimetric immunoenzymatic results, we found that the minimal detectable labelled DNA (MDLD) was 312 pg with the 5-FdUrd and thy - methods for 5-BrdUrd labelled plasmid DNA. 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy - and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA. Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA.

Apolipoprotein E alleles in Cuban patients with mild cognitive impairment.

Background: Apolipoprotein E (ApoE) ∊4 genotype is the most clearly documented risk factor for Alzheimer’s disease (AD). Epidemiological studies demonstrate an accelerated rate of progression to dementia and AD in patients with mild cognitive impairment (MCI). We assessed the ApoE allele and genotypes frequencies in Cuban patients with MCI. Methods: We performed ApoE genotyping of 74 Cuban patients more than 65 years old. Cognitive assessments included the Mini-Mental State Examination (MMSE) and a cognitive battery for evaluating memory, attention, perception, and executive function. Results: Cognitive impairments were characterized by amnesia and executive deficits in patients with MCI. The Apo ∊4 allele frequency was 0.196 in patients with MCI, 10-fold higher than that in the controls. Patients carrying the ∊4 allele exhibited poorer performance in MMSE and tests assessing executive function and short-term memory than noncarriers. Conclusions: The patients exhibited amnestic MCI multiple domains. Cognitive performance was worse in patients who carried the ApoE ∊4 allele.

PCR of Immobilized DNA Molecules Isolated from Human Blood Cells by a Non‐Enzymatic Method

Abstract DNA molecules suitable for amplification by Polymerase Chain Reaction were obtained by immobilizing whole blood or isolated leukocytes and incubating the immobilized cells for one hour with the known non‐enzymatic solution described for preparing intact DNA molecules for PFGE. Cell immobilization was done in agarose gels and punches of 1.2 mm of diameter had the amount of DNA needed for amplifying chromosomal and mitochondrial sequences, many times. The approach was successfully used in preparing DNA molecules from multiple samples in flat‐bottom 96‐well ELISA plates. The procedure is simple and does not demand special conditions for sample transportation or conservation; thus, it should be useful to collect and process samples under field conditions in epidemiological studies.

Algorithm to Predict MiniCHEF Electrophoresis Patterns of Entamoeba histolytica DNA

*Departamento de Biologia Molecular, Division de Neurociencias, Centro Nacional de Investigacion Cientifica, Havana, Cuba **Programa de Biomedicina Molecular, Centro de Investigacion en Ciencia Aplicada y Tecnologia Avanzada del I.P.N. (CICATA-IPN), Mexico City, Mexico ***Departamento de Patologia Experimental, ****Programa Multidisciplinario en Biomedicina Molecular, Centro de Investigacion y de Estudios Avanzados del I.P.N. (Cinvestav), Mexico City, Mexico

Telomeric Repeat-Binding Factor Homologs in Entamoeba histolytica: New Clues for Telomeric Research

Telomeric Repeat Binding Factors (TRFs) are architectural nuclear proteins with critical roles in telomere-length regulation, chromosome end protection and, fusion prevention, DNA damage detection, and senescence regulation. Entamoeba histolytica, the parasite responsible of human amoebiasis, harbors three homologs of human TRFs, based on sequence similarities to their Myb DNA binding domain. These proteins were dubbed EhTRF-like I, II and III. In this work, we revealed that EhTRF-like I and II share similarity with human TRF1, while EhTRF-like III shares similarity with human TRF2 by in silico approach. The analysis of ehtrf-like genes showed they are expressed differentially under basal culture conditions. We also studied the cellular localization of EhTRF-like I and III proteins using subcellular fractionation and western blot assays. EhTRF-like I and III proteins were enriched in the nuclear fraction, but they were also present in the cytoplasm. Indirect immunofluorescence showed that these proteins were located at the nuclear periphery co-localizing with Lamin B1 and trimethylated H4K20, which is a characteristic mark of heterochromatic regions and telomeres. We found by transmission electron microscopy that EhTRF-like III was located in regions of more condensed chromatin. Finally, EMSA assays showed that EhTRF-like III forms specific DNA-protein complexes with telomeric related sequences. Our data suggested that EhTRF-like proteins play a role in the maintenance of the chromosome ends in this parasite.

Identification of small nuclear DNA molecules of the cuban sugarcane cultivar Ja60-5 via pulsed field gel electrophoresis

We present evidence of the existence of at least 7 small nuclear DNA molecules in the Ja60-5 sugarcane cultivar genome. These small molecules were separated by means of transversal alternating field electrophoresis (TAFE); their sizes ranged from about 230–3000 kbp, and they hybridized with telomeric, subcentromeric, and various nuclear DNA probes. Here, we also report the use and the modifications of DNA from microorganisms, to immobilize nuclear DNA from sugarcane suitable for analysis via pulsed field gel electrophoresis. The protocol is simple, inexpensive, and easy to perform.