Widely Used Enhancer of Eukaryotic Expression Vectors Is Strongly and Differentially Regulated in Fibroblast, Myoblast, and Teratocarcinoma Cell Lines

The expression of transgenes in eukaryotic cells is a powerful approach in cell biology. In most cases, it is based on the activity of strong and constitutive viral cis-acting elements in eukaryotic expression vectors. Here we show that a widely used such element derived from an early gene of human cytomegalovirus is strongly and differentially regulated in mouse cell lines. We analyzed cytomegalovirus promoter-driven expression of stably transfected transgenes in growing, confluent, and differentiating mouse 3T3 fibroblasts, C2C12 myoblasts, and P19 teratocarcinoma cells. In the fibroblasts, transgene expression was strongly downregulated in confluent cultures and was upregulated in growing or confluent cultures by phorbol ester. In contrast, no downregulation by confluency, nor upregulation by phorbol ester, was detected in C2C12 cells. In addition, while marked upregulation was detected in differentiating myotubes, transgene expression was downregulated when differentiating teratocarcinoma cells assumed a neuronal phenotype. These results demonstrate the existence of drastic differences in the regulation of transgene expression in different types of cell lines, indicating that when studying transgene function in cells that are not growing exponentially, viral promoter-driven expression should not be taken for granted.     

Metabolic utilization of muscular L-proline in 24-hr starved rats.

1. The aim of this paper was to study the in vivo skeletal muscle L-proline related to its destination to other key tissues such as liver and intestine as well as to give some insight into the role of blood cells in proline handling. 2. L-U-[14C]Proline was injected intramuscularly and following by sampling of blood, liver, intestine and contralateral muscle at 20 and 30 min after injection. 3. The distribution of radioactivity between blood cells and plasma and in total and individual amino acids, protein and glycogen fractions was determined in the above tissues. 4. The pattern of well fed rats was compared with those submitted to 24-hr complete starvation. 5. During starvation a minor degree of proline oxidation occurs. 6. The main destruction of proline in the liver seem to be the synthesis of proteins. 7. The radioactivity recovered in the blood proline fraction of starved rats is twice that of the fed rats and that it could be attributed mainly to plasma protein. 8. We have obtained in vivo evidence for the role of erythrocyte in the interorgan proline transport.     

Human toxicology of BCG applied in cancer immunotherapy

Summary Seventy-five patients were treated for short periods with BCG either per os (7), by aerosols (2), i.d. with needles, i.d. with heaf-gun, on one or four scarification areas, i.v., or intratumorally. Two hundred and seventy-seven were treated for more than 3 years by BCG applied scarifications.The local reactions after application on scarification are negligible, the most frequent being pruritus and adenopathies.The systemic reactions are due to the BCG septicemia which is induced and which has been proved by the search for liver granulomas. Some reactions are of an allergic nature (e.g., choroiditis), but most are direct manifestations of the septicemia (fever, hepatomegaly, splenomegaly). One death was observed after tumoral injection in a terminal patient, otherwise there were no deaths, even in the patients under long-term treatment with the other metabolites.Deterioration of immune reactions may be induced either by the method of BCG application which is followed by a (probably) very small penetration (on scarified area in allergics), or by the penetration of high doses (four scarified areas in anergics and intravenous injections in anergics and in allergics). Reprint requests should be addressed to: G. Mathé, 14-16, avenue Paul-Vaillant-Couturier, F-94800 Villejuif (France)     

A high density physical map of chromosome 1BL supports evolutionary studies,map-based cloning and sequencing in wheat

Background

As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.

Results

Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.

Conclusions

Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.     

Cytotaxonomy and geographic distribution of cytotypes of species of the South American genus Chrysolaena (Vernonieae,Asteraceae)

Understanding speciation and biodiversity patterns in plants requires knowledge of the general role of climate in allowing polyploids to escape competition and persist with their diploid progenitors. This is a particularly interesting issue in widespread species that present multiple ploidy levels and occur across a heterogeneous environment. Chrysolaena (Vernonieae, Asteraceae) is a cytogenetically very diverse genus, with significant interspecific and intraspecific ploidy level variation and with continuous distribution across South America. No previous studies have summarized chromosome count data of Chrysolaena or addressed the cytogeography of the genus. Ploidy level of Chrysolaena species was determined by chromosome counting during mitosis and/or meiosis; the geographic distribution of cytotypes was examined and the correlations between the distribution of particular cytotypes and current ecological conditions were evaluated. A total of 43 new chromosome counts and five ploidy levels (2x, 4x, 6x, 7x, 8x) were reported. The chromosome number of C. cordifolia (2n = 7x = 70) and a new cytotype for C. propinqua var. canescens (2n = 4x = 40) are reported for the first time. Three geographic areas with high diversity of cytotypes and species were detected. The results obtained do not suggest a clear distribution pattern that depends on climatic factors for Chrysolaena populations. However, a geographic pattern was identified in the distribution of ploidy levels, with diploid species presenting a more restricted distribution than polyploid species.     

Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.     

Evaluation and classification of RING-finger domains encoded by the <Emphasis Type="Italic">Arabidopsis</Emphasis> genome

Background

In computational analysis, the RING-finger domain is one of the most frequently detected domains in the Arabidopsis proteome. In fact, it is more abundant in Arabidopsis than in other eukaryotic genomes. However, computational analysis might classify ambiguous domains of the closely related PHD and LIM motifs as RING domains by mistake. Thus, we set out to define an ordered set of Arabidopsis RING domains by evaluating predicted domains on the basis of recent structural data.

Results

Inspection of the proteome with a current InterPro release predicts 446 RING domains. We evaluated each detected domain and as a result eliminated 59 false positives. The remaining 387 domains were grouped by cluster analysis and according to their metal-ligand arrangement. We further defined novel patterns for additional computational analyses of the proteome. They were based on recent structural data that enable discrimination between the related RING, PHD and LIM domains. These patterns allow us to predict with different degrees of certainty whether a particular domain is indeed likely to form a RING finger.

Conclusions

In summary, 387 domains have a significant potential to form a RING-type cross-brace structure. Many of these RING domains overlap with predicted PHD domains; however, the RING domain signature mostly prevails. Thus, the abundance of PHD domains in Arabidopsis has been significantly overestimated. Cluster analysis of the RING domains defines groups of proteins, which frequently show significant similarity outside the RING domain. These groups document a common evolutionary origin of their members and potentially represent genes of overlapping functionality.