Regulation of nitrate reductase of Neurospora at the level of transcription and translation

The presence of nitrate is required for the induced synthesis of NADPH-nitrate reductase and its related partial activity Benzyl Viologen-nitrate reductase in a wild-type strain of Neurospora. In nit-3, a mutant lacking complete NADPH-nitrate reductase activity but retaining the partial activity Benzyl Viologen-nitrate reductase, the presence of nitrate ions is not required for the de-repressed synthesis of the latter enzyme. The accumulation of the capacity to synthesize nitrate reductase, and the related Benzyl Viologen-nitrate reductase, in the absence of protein synthesis does not require nitrate in the normal strain or in strain nit-3. Ammonia antagonizes the accumulation of this capacity in both strains. Nitrate is required for the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain. Nitrate is not required for the comparable function in strain nit-3. Ammonia appears to stop the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain and in strain nit-3. The effects of nitrate, or ammonia and of no nitrogen source on the induced synthesis of nitrate reductase cannot be explained on the basis of the effects of the different nitrogen sources on general synthesis of RNA or of protein.     

Detection of adaptive response at chromosome level.

We have just started the basic study to detect the genetic alterations at chromosome level as a result of radioadaptive response. The assay system is based upon the analysis of loss of heterozygosity (LOH) induced in human lymphoblastoid cell TK6, which were pre-irradiated with low-doses of ionizing radiation (IR) before the challenging irradiation. In our previous study, this analysis was shown to be very sensitive to IR because the radiation-specific hemizygous LOHs (interstitial deletions) were observed after 10 cGy of IR (X-rays or accelerated carbon-ion beam). Here, we would like to introduce our plan how to detect the changes in such radiation-specific LOH patterns by the pre-irradiation of TK6. If we succeed the detection, the radioadaptation assay system can be used for elucidating the biological effects of low-doses of space ionizing radiation. In addition, we are also considering the modification of assay system by introducing the site-specific chromosome breakage (DNA double-strand break) instead of challenging IR. Furthermore, the preliminary results of the experiments using frozen TK6 cells for the preparation of ISS experiments.     

Tight,cell type-specific control of LNX expression in the nervous system,at the level of transcription,translation and protein stability

LNX1 and LNX2 are E3 ubiquitin ligases that can interact with Numb — a key regulator of neurogenesis and neuronal differentiation. LNX1 can target Numb for proteasomal degradation, and Lnx mRNAs are prominently expressed in the nervous system, suggesting that LNX proteins play a role in neural development. This hypothesis remains unproven, however, largely because LNX proteins are present at very low levels in vivo. Here, we demonstrate expression of both LNX1 and LNX2 proteins in the brain for the first time. We clarify the cell-type specific expression of LNX isoforms in both the CNS and PNS, and identify a novel LNX1 isoform. Using luciferase reporter assays, we show that the 5′ untranslated region of the Lnx1_variant 2 mRNA, that generates the LNX1p70 isoform, strongly suppresses protein production. This effect is mediated in part by the presence of upstream open reading frames (uORFs), but also by a sequence element that decreases both mRNA levels and translational efficiency. By contrast, uORFs do not negatively regulate LNX1p80 or LNX2 expression. Instead, we find some evidence that protein turnover via proteasomal degradation may influence LNX1p80 levels in cells. These observations provide plausible explanations for the low levels of LNX1 proteins detected in vivo.     

CSDBase: an interactive database for cold shock domain-containing proteins and the bacterial cold shock response

CSDBase (http://www.chemie.uni-marburg.de/~csdbase/) is an interactive Internet-embedded research platform providing detailed information on proteins containing the cold shock domain (CSD). It consists of two separated database cores, one dedicated to CSD protein information, and one to provide a powerful resource to relevant literature with emphasis on the bacterial cold shock response. In addition to detailed protein information and useful cross links to other web sites, CSDBase contains computer-generated CSD structure models for most CSD-containing protein sequences available at NCBI non-redundant protein database at the time of CSDBase establishment. These models were calculated on the basis of known crystal and/or NMR structures using SWISS-MODEL and can be downloaded as PDB structure coordinate files for viewing and for manipulation with other software tools. CSDBase will be regularly updated and is organized in a compact form providing user friendly interfaces to both database cores which allow for easy data retrieval.     

A method for comparing DNA replication programmes at the level of the chromosome bands

During DNA synthesis, each pair of homologous chromosomes replicates its bands in a precise order and at a specific time. When using asynchronous cell populations, this replication programme has to be reconstructed from a series of "stills"--serial samples taken at intervals through S-transit. Obviously, the result obtained is dependent upon the kinetic progression of cells through the cycle, and any perturbation of the cycle. A difficulty arises when we wish to compare the replication programme of a chromosome in two different cultures (e.g. cells from different origins, or after different treatments). Kinetic differences between cultures make it almost impossible to obtain two samples for analysis containing the same "mixture" of cells. Thus, a false programme difference could be introduced, or a real one masked. In this paper, we present a method of comparison that overcomes this problem. It is based upon the observation that with serial sampling of steady-state cell populations through S-phase, band appearance curves are sigmoidal and are very well approximated by cumulative Normal distributions with very similar standard deviations. If a family of such curves, closely spaced in time, is sampled twice, the two observed frequencies for each curve are related, their probits all lying on a single straight line. This line has a slope of 45 degrees and its displacement from the origin is a function of the time interval between the two samples. Given two identical families of such curves, exactly the same relationship will hold if one sample is drawn from each. If, however, the two families differ (in order, spacing, standard deviation etc.), the probit/probit plot will deviate in various ways from a straight line with 45 degrees slope. Any two subsets of chromosome replication-band frequencies can be regarded as derived from a family of cumulative Normal curves and probit/probit comparisons used to test the similarity of their replication programmes.