Detection of DNA polymerase λ activity during seed germination and enhancement after salinity stress and dehydration in the plumules of indica rice (Oryza sativa L.).

DNA polymerase λ (DNA pol λ) is the only reported X-family DNA polymerases in plants and has been shown to play a significant role in dry quiescent seeds, growth, development and nuclear DNA repair. cDNA for DNA pol λ has been reported in Arabidopsis and japonica rice cultivar and has been characterized from E. coli expressed protein, but very little is known about its activity at protein level in plants. The enzymatic activity of DNA pol λ was studied in dry, imbibed and during different germination stages of indica rice IR-8 (salt sensitive) by in-gel activity assay to determine its physiological role in important stages of growth and development. The upstream sequence was also analyzed using plantCARE database and was found to contain several cis-acting elements, including light responsive elements, dehydration responsive elements, Myb binding sites, etc. Hence, 4-day-old germinating seedlings of IR29, a salt-sensitive, but high yielding indica rice cultivar and Nonabokra, a salt-tolerant, but low yielding cultivar were treated with water (control) or 250 mM NaCl or 20% polyethyleneglycol-6000 for 4 and 8 h. The protein was analyzed by in vitro DNA pol λ activity assay, in-gel activity assay and Western blot analysis. DNA pol λ was not detected in dry seeds, but enhanced after imbibition and detectable from low level to high level during subsequent germination steps. Both salinity and dehydration stress led to the enhancement of the activity and protein level of DNA pol λ, as compared to control tissues. This is the first evidence of the salinity or dehydration stress induced enhancement of DNA pol λ activity in the plumules of rice (Oryza sativa L.) cultivars.

Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.

PURPOSE: To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens. METHODS: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. RESULTS: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. CONCLUSIONS: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

Análisis molecular de la hemofilia A en familias del noroeste de México / molecular analysis of Hemophilia A in northeastern mexican families

Empleando las técnicas de Southern para la detección de genes por hibridación con una sonda radiactiva de la reacción en cadena de la polimerasa para la amplificación de fragmentos génicos, se practicó el análisis molecular a nivel de ADN para detectar portadoras de la hemofilia A. Este se basó en el estudio del polimorfismo para la enzima de restricción Bcl I presente en el intró 18 del gen para el factor VIII de la coagulación. Se analizaron ocho familias de noroeste de México con antecedentes de la enfermedad, que comprendieron un total de 43 individuos. De las 27 mujeres que formaban parte de la muestra, 17 requerían el diagnóstico de portadora, el cual se estableció en tres de ellas, se excluyó en cinco y no se pudo determinar en nueve. Las frecuencias encontradas en la muestra para el polimorfismo fueron de 63 por ciento para el alelo de tamaño y de 37 por ciento para el alelo de tamaño menor, determinando un porcentaje de mujeres heterocigotas (informativas) de 48.2 por ciento y fijando en este valor la utilidad teórica del polimorfismo en las familias aquí incluidas. Estas frecuencias muestran simulitud con las de las poblaciones mediterráneas y establecen que la utilidad del polimorfismo es mayor en nuestra muestra que en otros grupos étnicos.