Iterative in vivo assembly of large and complex transgenes by combining the activities of φC31 integrase and Cre recombinase

We have used the C31 integrase to introduce large DNA sequences into a vertebrate genome and measure the efficiency of integration of intact DNA as a function of insert size. Inserts of 110 kb and 140 kb in length may be integrated with about 25% and 10% efficiency respectively. In order to overcome the problems of constructing transgenes longer than ~150 kb we have established a method that we call; ‘Iterative Site Specific Integration’ (ISSI). ISSI combines the activities of C31 integrase and Cre recombinase to enable the iterative and serial integration of transgenic DNA sequences. In principle the procedure may be repeated an arbitrary number of times and thereby allow the integration of tracts of DNA many hundreds of kilobase pairs long. In practice it may be limited by the time needed to check the accuracy of integration at each step of the procedure. We describe two ISSI experiments, in one of which we have constructed a complex array of vertebrate centromeric sequences of 150 kb in size. The principle that underlies ISSI is applicable to transgenesis in all organisms. ISSI may thus facilitate the reconstitution of biosynthetic pathways encoded by many different genes in transgenic plants, the assembly of large vertebrate loci as transgenes and the synthesis of complete genomes in bacteria.     

Preliminary evaluation of thyroid C cells in rats with chronic renal failure

Numerous water-electrolyte and hormonal disturbances, including secondary hyperparathyroidism, occur in the course of chronic renal failure. It is assumed that chronic renal failure should affect the activity of C cells in the thyroid gland. Thus, the aim of the study was to evaluate immunohistochemically thyroid C cells in rats with experimental uremia. 30 days after nephrectomy, thyroid fragments were collected from experimental rats. Paraffin embedded sections were stained with H+E and by silver impregnation. Calcitonin (CT), synaptophysin (SY), somatostatin (ST), and neuron-specific enolase (NSE) were detected immunohistochemically in the C cells. A very distinct increase in C cell number in the thyroid and the weakening of majority of examined reactions were observed in rats with experimental uremia.     

The dynamics of genome replication using deep sequencing

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology.     

Immunoreactivity of neuroendocrine cells in the respiratory tract in rats with experimental uremia after thyroparathyroidectomy

Animals with experimental uremia, which underwent thyroparathyroidectomy, reveal numerous metabolic disorders that can influence morphology and activity of endocrine cells of the scattered neuroendocrine system. The aim of the study was the evaluation of the influence of thyroparathyroidectomy in rats with chronic renal failure on APUD system cells localized in the respiratory tract. The examination was conducted on the group of 20 rats. Thyroparathyroidectomy was performed 30 days after nephrectomy. Fragments of the lungs and trachea were collected 14 days after the operation. Routinely prepared paraffin sections were stained with H+E and with silver method. The immunohistochemical reactions were conducted with the use of antibodies against calcitonin (CT), synaptophysin (SPh), somatostatine (ST), and neuron-specific enolase (NSE) The results were estimated in light microscope on the basis of stain reaction of endocrine cells. Our examination showed that chronic renal failure affects the functioning of endocrine cells. We also observed the increase in APUD system cell number in the trachea and the lungs after thyroparathyroidectomy in uremic rats.     

Micropropagation of Allium wallichii kunth, a threatened medicinal plant of Nepal

Summary Bulbs and aerial parts of the Nepalese plant Allium wallichii are widely used for medicinal purposes and as a spice. Due to overharvesting the natural populations of the species have been increasingly reduced and the domestication of the species should be considered. For the purpose of the production of plantlets suitable for field culture, a micropropagation procedure based on multiple shoot culture has been established. Multiplication factors of 4.6 on average were possible on MS medium supplemented with 20 μM zeatin. After rooting on MS medium with 10 μM indolebutyric acid, plantlets were acclimatized to greenhouse conditions and transferred to the field with good success. Part of the PhD thesis of P. R. Malla.     

The dynamics of morphological changes in the pyloric endocrine cells of rats with uremia

Disturbances in renal homeostatic function lead to changes in endocrine cell secretory activity. The aim of this study was the histomorphological estimation of dependence of gastric APUD system cell morphology and function on the time after subtotal nephrectomy in Wistar rats. Fragments of gastric pylorus were collected 1. 2, 4, and 6 weeks after nephrectomy. Paraffin sections were stained with H+E and by silver impregnation. Immunohistochemical reactions with the use of specific antibodies against calcitonin gene-related peptide (CGRP), synaptophysin (SPh). somatostatin (ST), and neuron-specific enolase (NSE) were also performed. Immunoreactivity of the examined substances in the pyloric mucosa in the first week after nephrectomy was lower than in the control group. However, in the following time intervals, endocrine cells showed stronger immunostaining in comparison with the control rats. The results suggest that chronic renal failure can modulate secretory activity of APUD system cells.     

Anti-trichomonal IgA antibodies in trichomoniasis before and after treatment

Anti-trichomonal IgA antibodies were estimated by enzyme-linked immunosorbent assay (ELISA) in serum and vaginal secretions of 25 symptomatic and 25 asymptomaticTrichomonas vaginalis positive patients before and after treatment and in 25 age-matched controls. Significantly higher levels of antitrichomonad IgA antibodies were found inT. vaginalis positive patients when compared to control subjects, especially in vaginal secretions. In addition, a significant decrease in these antibodies was observed after treatment, which was more pronounced in vaginal secretions. It seems that anti-trichomonal IgA antibodies in serum and more so in vaginal secretions are directly related to and specific to the presence ofT. vaginalis in the urogenital tract.