Towards quantitative in situ hybridization

In situ hybridization analysis of tissue mRNA concentrations remains to be accepted as a quantitative technique, even though exposure of tissue sections to photographic emulsion is equivalent to Northern blot analysis. Because of the biological importance of in situ quantification of RNA sequences within a morphological context, we evaluated the quantitative aspects of this technique. In calibrated microscopic samples, autoradiographic signal (density of silver grains) was proportionate to the radioactivity present, to the exposure time, and to time of development of the photographic emulsion. Similar results were obtained with tissue sections, showing that all steps of the in situ hybridization protocol, before and including the detection of the signal, can be reproducibly performed. Furthermore, the integrated density of silver grains produced in liver and intestinal sections by the in situ hybridization procedure using 35S-labeled riboprobes is directly proportionate to the signal obtained by quantitative Northern blot analysis. The significance of this finding is that in situ quantification of RNA can be realized with high sensitivity and with the additional advantage of the possibility of localizing mRNA within the cells of interest. Application of this procedure on fetal and adult intestinal tissue showed that the carbamoylphosphate synthetase (CPS)-expressing epithelial cells of both tissues accumulated CPS mRNA to the same level but that whole-organ CPS mRNA levels decreased four-to fivefold in the same period, owing to a comparable decrease in the number of CPS-expressing cells in total intestinal tissu

Anomalous Power Law Distribution of Total Lifetimes of Branching Processes Relevant to Earthquakes

We consider a branching model of triggered seismicity, the ETAS (epidemic-type aftershock sequence) model which assumes that each earthquake can trigger other earthquakes (``aftershocks''). An aftershock sequence results in this model from the cascade of aftershocks of each past earthquake. Due to the large fluctuations of the number of aftershocks triggered directly by any earthquake (``productivity'' or ``fertility''), there is a large variability of the total number of aftershocks from one sequence to another, for the same mainshock magnitude. We study the regime where the distribution of fertilities $\mu$ is characterized by a power law $\sim 1/\mu^{1+\gamma}$ and the bare Omori law for the memory of previous triggering mothers decays slowly as $\sim 1/t^{1+\theta}$, with $0 < \theta <1$ relevant for earthquakes. Using the tool of generating probability functions and a quasistatic approximation which is shown to be exact asymptotically for large durations, we show that the density distribution of total aftershock lifetimes scales as $\sim 1/t^{1+\theta/\gamma}$ when the average branching ratio is critical ($n=1$). The coefficient $1<\gamma = b/\alpha<2$ quantifies the interplay between the exponent $b \approx 1$ of the Gutenberg-Richter magnitude distribution $\sim 10^{-bm}$ and the increase $\sim 10^{\alpha m}$ of the number of aftershocks with the mainshock magnitude $m$ (productivity) with $\alpha \approx 0.8$. More generally, our results apply to any stochastic branching process with a power-law distribution of offsprings per mother and a long memory.Comment: 16 pages + 4 figure

Marked differences in tissue-specific expression of chitinases in mouse and man

FWN – Publicaties zonder aanstelling Universiteit Leide

Organ-specific activity of the 5' regulatory region of the glutamine synthetase gene in developing mice

Glutamine synthetase (GS) converts ammonia and glutamate into glutamine. We assessed the activity of the 5' regulatory region of the GS gene in developing transgenic mice carrying the chloramphenicol acetyltransferase (CAT) gene under the control of 3150 bp of the upstream sequence of the rat GS gene to obtain insight into the spatiotemporal regulation of its pattern of expression. To determine the organ-specific activity of the 5' regulatory region CAT and GS mRNA expression were compared by ribonuclease-protection and semi-quantitative in situ hybridization analyses. Three patterns were observed: the 5' region is active and involved in the regulation of GS expression throughout development (pericentral hepatocytes, intestines and epididymis); the 5' region shows no activity at any of the ages investigated (periportal hepatocytes and white adipose tissue); and the activity of the 5' region becomes repressed during development (stomach, muscle, brown adipose tissue, kidney, lung and testis). In the second group, an additional element must be responsible for the activation of GS expression. The last group included organs in which the 5' regulatory region is active, but not in the cells that express GS. In these organs, the activity of the 5' regulatory region must be repressed by other regulatory regions of the GS gene that are missing from the transgenic construct. These findings indicate that in addition to the 5' regulatory region, at least two unidentified elements are involved in the spatiotemporal pattern of expression of G

Developmental changes in rat cardiac DNA, RNA and protein tissue base: implications for the interpretation of changes in gene expression

During cardiac development the expression levels of many genes change as determined by Northern blot, dot blot, RNase protection, quantitative RT-PCR. Western blot or immunoprecipitation analyses. It is not always realized that the total amount of RNA or protein per gram of heart, dubbed tissue base, may change significantly during development as well. If this would be the case, this has to be taken into account. So far, the (changing) tissue base has not been established during cardiac development. To this end developmental profiles of cardiac DNA, RNA and protein concentration were determined in rats ranging in age from embryonic day 13 until neonatal day 121. The profiles show significant development changes in each parameter, that closely match the distinct growth phases of the developing heart and provide the parameters that are essential for an adequate interpretation of changes in the amount of a distinct mRNA and/or protein. In a comparison between in situ hybridization and Northern blot analysis it is demonstrated that the same developmental profile leads to an almost opposite conclusion depending on whether or not the changing tissue base is taken into account. These findings are of great interest for studies aimed at unravelling the molecular mechanisms underlying the regulation of gene expression during cardiac developmen