Ultraviolet-B exposure leads to up-regulation of senescence-associated genes in Arabidopsis thaliana.

Exposure to UV-B radiation resulted in a loss of chlorophyll and an increase in lipid damage in a similar manner to that induced during natural senescence. In addition, exposure to UV-B led to the induction of a number of genes associated with senescence (SAG12, 13, 14, and 17). These results show, for the first time, that exposure to UV-B can lead to cellular decline through active and regulated processes involving many genes also associated with natural senescence.

Gene expression profiling of cancer by use of DNA arrays: how far from the clinic?

DNA arrays allow the simultaneous analysis of expression levels for thousands of genes in normal and pathological tissues and hold great promise in molecular medicine, notably in cancer research. The great biological and clinical diversity present in human tumours is poorly characterised by the current classification systems. DNA arrays can provide a better understanding of oncogenesis, leading to improvements in cancer management. First, the identification of new target genes and pathways will allow the development of specific molecular-based anticancer drugs. Secondly, expression profiles will permit tumour classification in more homogeneous diagnostic and prognostic groups, as well as the identification of new clinically and biologically relevant tumour subclasses. Here, we review the technology and present some cancer studies with promising results. Finally, we discuss some of the issues that must be resolved in the near future, so that DNA arrays can fulfil the aims mentioned above.

The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9.

Chemokines are key regulators of migration in lymphoid tissues. In the thymus, maturing thymocytes move from the outer capsule to the inner medulla and thereby interact with different types of stromal cells that control their maturation and selection. In the process of searching for molecules specifically expressed at different stages of mouse thymic differentiation, we have characterized the cDNA coding for the thymus-expressed chemokine (TECK) and its receptor CCR9. The TECK receptor gene was isolated and shown to be localized on the mouse chromosome 9F1-F4. Thymic dendritic cells have been initially thought to be a prevalent source of TECK. In contrast, our results indicate that thymic epithelial cells constitute the predominant source of TECK. Consistent with the latter distribution, the TECK receptor is highly expressed by double-positive thymocytes, and TECK can chemoattract both double-positive and single-positive thymocytes. The TECK transcript is also abundantly expressed in the epithelial cells lining the small intestine. In conclusion, the interplay of TECK and its receptor CCR9 is likely to have a significant role in the recruitment of developing thymocytes to discrete compartments of the thymus.

Expression profiling: DNA arrays in many guises.

DNA arrays have become the preferred method for large-scale expression measurement. Such data are needed in view of the large amounts of sequence data available: expression levels in a number of different tissues or situations provide a first step toward functional characterisation of new entities revealed by DNA sequencing. Although the basic principle of measurement is in all cases based on hybridisation of a mixed probe derived from tissue RNA to large sets of DNA fragments representing many genes, a number of different forms of implementation of this principle are at hand. They are briefly described and compared, emphasizing the important issue of sensitivity and sample requirements and the accessibility of the methods to academic scientists. When these factors are taken into account, it appears that, contrary to a largely prevalent impression, the "best" approach is not necessarily always provided by the widely advertised glass microarrays or oligonucleotide chips.

Sensitivity issues in DNA array-based expression measurements and performance of nylon microarrays for small samples.

DNA or oligonucleotide arrays are widely used for large-scale expression measurements, using various implementations: macroarrays in which DNA is spotted onto nylon membranes of relatively large dimensions (with radioactive detection) on the one hand; microarrays on glass slides and oligonucleotide chips, both used with fluorescent probes, on the other hand. Nylon micro-arrays with colourimetric detection have also been described recently. The small physical dimensions of miniaturized systems allow small hybridization volumes (2-100 microl) and provide high probe concentrations, in contrast to macroarrays. We show, however, that actual sensitivity (defined as the amount of sample necessary for detection of a given mRNA species) is in fact similar for all these systems and that this is mostly due to the very different amounts of target material present on the respective arrays. We then demonstrate that the combination of nylon microarrays with(33)P-labelled radioactive probes provides 100-fold better sensitivity, making it possible to perform expression profiling experiments using submicrogram amounts of unamplified total RNA from small biological samples. This has important implications in basic and clinical research and makes this alternative approach particularly suitable for groups operating in an academic context.

'Genomics': buzzword or reality?

'Genomics' has become a widely used term, covering a range of approaches that make use of the newly acquired wealth of genome data (both on man and on a number of model organisms) to gain new insights and accelerate research. This review attempts to present a clear and balanced view of developments in this field, to describe the four major approaches that contribute to genomics (bioinformatics, genetic analysis of extended populations, large-scale expression studies, functional approaches), and to indicate applications in basic and pharmaceutical research.

Engagement of natural cytotoxicity programs regulates AP-1 expression in the NKL human NK cell line.

NK cell cytotoxicity is a fast and efficient mechanism of target cell lysis. Using transcription analysis, such as multiplex messenger assays, we show here that natural cytotoxicity exerted by the human NKL cell line correlates with mRNA accumulation of very early activator protein (AP)-1 transcription factor genes such as JunB, FosB and c-Fos. In addition, DNA-binding activities of Jun-Fos heterodimers were observed by electrophoretic mobility shift assays during the course of natural cytotoxicity. Interaction between immunoglobulin-like transcript-2/leukocyte Ig-like receptor 1 on NKL cells and HLA-B27 on target cells leads to an impairment of NKL natural cytotoxicity, which correlates with an absence of JunB, FosB, and c-Fos transcription, as well as an absence of their DNA-binding activity. Our studies thus indicate that, despite the rapidity of NK cell-mediated lysis, AP-1 transcription factor is activated during the early stage of NK cell cytolytic programs and that engagement of NK cell inhibitory receptors for MHC class I molecules impairs the very early activation of AP-1.

'LABNOTE', a laboratory notebook system designed for academic genomics groups.

We have developed a relational laboratory database system, adapted to the daily book-keeping needs of laboratories that must keep track of information acquired on hundreds or thousands of clones in an effective and user-friendly fashion. Data, whether final or related to experiments in progress, can be accessed in many different ways, e.g. by clone name, by gene, by experiment or through DNA sequence. Updating, import and export of results is made easier by specially developed tools. This system, in network version, serves several groups in our Institute and (over the Internet) elsewhere, and is instrumental in collaborative studies based on expression profiling. It can be used in many similar situations involving progressiveaccumulation of information on sets of clones or related objects.

Differential gene expression in CD3epsilon- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation.

A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3epsilon- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrbeta, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function.

Factors affecting UV-B-induced changes in Arabidopsis thaliana L. gene expression: the role of development, protective pigments and the chloroplast signal.

Gene expression is known to change in response to UV-B radiation. In this paper, we have investigated three factors in Arabidopsis leaves that are likely to influence these changes: development, protective pigments and the 'chloroplast signal'. During late leaf development the major change in pigment composition, after exposure to UV-B radiation, is an increase in UV-absorbing pigments. Chl and Chl a/b ratio do not change substantially. Similarly Chl fluorescence is not altered. In contrast, RNA transcripts for photosynthetic proteins are reduced more in older leaves than in young leaves. To determine the role of flavonoids in UV-B protection, plants of Arabidopsis mutant tt-5, which have reduced flavonoids and sinapic esters, were exposed to UV-B and RNA transcript levels determined. The tt-mutants were more sensitive to UV-B radiation than wild-type. To examine the role of the chloroplast signal in regulating UV-B-induced changes in gene expression, Arabidopsis gun mutants (genome uncoupled) have been used. The results show that UV-B-induced down-regulation still takes place in gun mutants and strongly suggests that the chloroplast signal is not required. Overall, this study clearly demonstrates that UV-B-induced changes in gene expression are influenced by both developmental and cellular factors but not chloroplastic factors.

Large-scale expression measurement by hybridization methods: from high-density membranes to "DNA chips".

The vast amount of sequence information becoming available on genes from man and from other species calls for corresponding increases in the rate of collection for data of a more functional nature. Expression measurements often constitute a first step in this direction, and can be performed on a reasonably large scale using highly parallel hybridization methods. Large sets of targets (clones, inserts, oligonucleotides) are hybridized with labeled complex probes prepared from total cell or organ mRNA; under the proper conditions, signals measure the relative abundance of each sequence species, and can be acquired quantitatively. These techniques are presently available in three formats: high-density membranes to be hybridized with radioactive complex probes, microarrays of DNA spots (a miniaturized version of the former technique) using fluorescent complex probes, and oligonucleotide chips that, although developed originally for mutation detection, can be adapted to perform expression measurements. The miniaturized formats clearly represent the future, since they allow higher sensitivity, assay of large numbers of entities and hopefully provide the opportunity to use small amounts of starting material.

Molecular cloning, sequencing and expression of the mRNA encoding human Cdx1 and Cdx2 homeobox. Down-regulation of Cdx1 and Cdx2 mRNA expression during colorectal carcinogenesis.

Defining the molecular mechanisms involved in cancer formation and progression is still a major challenge in colorectal-cancer research. Our strategy was to characterize genes whose expression is altered during colorectal carcinogenesis. To this end, the phenotype of a colorectal tumour was previously established by partial sequencing of a large number of its transcripts and the genes of interest were selected by differential screening on high-density filters with mRNA of colorectal cancer and normal adjacent mucosa. Fifty-one clones were found over-expressed and 23 were underexpressed in the colorectal-cancer tissues of the 5 analyzed patients. Among the latter, clones 6G2 and 32D6 were found of particular interest, since they had significant homology with several homeodomain-containing genes. The highest degree of similarity was with the murine Cdx1 for 6G2, and with the murine Cdx2 and hamster Cdx3 for 32D6. Using a RT-PCR approach, complete sequence of both types of homeobox-containing cDNA was obtained. The amino-acid sequence of the human Cdx1 is 85% identical to the mouse protein, and human Cdx2 has 94% identity with the mouse Cdx2 and hamster Cdx3. Tissue-distribution analysis of Cdx1 and Cdx2 mRNA showed that both transcripts were specifically expressed in small intestine, in colon and rectum. Twelve tissue samples from colorectal adenocarcinomas and the corresponding normal mucosa were analyzed by Northern blot. Expression of the 2 types of mRNA was either reduced or absent in 10 of them. Several colon-cancer cell lines were also analyzed. Cdx2 mRNA was absent from LS174T cells and Cdx1 mRNA was absent in PF11, TC7 and SW480 cells; none was detected in HT29 cells. It was concluded that decrease in human Cdx1 and/or Cdx2 expression is associated with colorectal tumorigenesis.

Modulation of mRNA levels in the presence of thymocytes and genome mapping for a set of genes expressed in mouse thymic epithelial cells.

Modulation of gene expression in mouse thymic epithelium upon culture in the presence of thymocytes (coculture) was studied by comparison of hybridization signatures on a set of nearly 5000 mouse thymus cDNA clones. Forty-nine differentially expressed clones (usually down-regulated in coculture) were characterized by tag sequencing. Many of them corresponded to entities that had not been described previously in the mouse, and were further characterized by genome mapping. This set of genes appears to be involved in growth regulation and differentiation within the thymus.

Differential mRNA expression in untreated and TNF-alpha elicited murine dendritic cells precursors.

We have compared the pattern of gene expression in long term cultured precursor dendritic cells (DC), either untreated (immature) or cultured for two days in the presence of recombinant murine (rm)-TNF alpha (mature). The hybridization signature of complex cDNA probes prepared from total RNA extracted from immature and mature DC were analyzed using a mouse thymic cDNA library, gridded on high density filters. For each clone spotted on the filters, we have measured using an imaging plate device the hybridization signals of the complex probe obtained from immature or mature DC. Comparative analysis of these values allowed us to identify differentially expressed gene products. Our goal is to identify a new set of genes induced or repressed during DC maturation elicited by rmTNF alpha treatment.

Multiplex messenger assay: simultaneous, quantitative measurement of expression of many genes in the context of T cell activation.

The hybridization signature approach, using colony filters and labeled complex probes, can provide high throughput measurement of gene activity. We describe here the implementation of this method to follow the expression levels of 47 genes in resting and activated T cells, as well as in epithelial cells. Using 4-fold spotting of colonies, imaging plate detection and various correction and normalization procedures, the technique is sensitive enough to quantify expression levels for sequences present at 0.005% abundance in the probe. Comparison with Northern blotting shows good consistency between the two methods. Upon activation of a T cell clone by an anti-CD3 antibody variations ranging from 2- to 20-fold are measured, some of which had not been reported previously. This 'multiplex messenger assay' method, performed using available commercial apparatus, can be used in many cases where simultaneous assessment of mRNA levels for many genes is of interest.

From hybridization image to numerical values: a practical, high throughput quantification system for high density filter hybridizations.

Hybridization to sets of bacterial colonies or PCR products arrayed on high density filters is used in a number of experimental schemes. In many cases it is desirable to collect quantitative information ('hybridization signatures') rather than indications on 'positive' and 'negative' colonies. We present a practical system, based on an imaging plate analyser and a customized version of commercial software, that makes such quantification feasible, and define its performance in terms of reproducibility and linearity. The system is far superior to methods based on autoradiography and should be useful in many projects that involve the increasingly popular high density filter format.

Differential gene expression in the murine thymus assayed by quantitative hybridization of arrayed cDNA clones.

High-throughput measurement of hybridization signatures obtained using complex probes prepared from poly(A)+ RNA and high-density cDNA colony filters is described. The performance of the system, elimination of artifacts, and verification of the validity of the data are discussed. cDNAs corresponding to sequences present at levels of approximately 0.01% in the complex probe can be detected. Good correlation is observed between expression profiles determined by this method and by Northern blotting. The method is applied to a preliminary investigation of differential expression in three cell types present in the murine thymus.

Genome programmes: an opportunity for biology.

The development of human molecular genetics in the eighties led to the implementation of projects aimed at a systematic study of the human genetic make-up--the so-called "Genome Projects". These have so far been mainly concerned with genome mapping, both by family studies using polymorphic markers (genetic mapping) and by direct analysis of DNA with suitable fractionation and cloning methods (physical mapping). Major progress has been made in both fields recently. Sequencing per se has remained limited because of technical constraints, although systematic cDNA sequencing has caught on in a large way. National policies are quite diverse, with the USA, the UK, France and Japan being the major players; funding sources range from governments to foundations. Many of the results, methods and concepts obtained or implemented during genome studies can be of great use to "conventional" laboratories, and efficient interfacing between these two worlds is an increasingly important issue.

Cloning and sequence analysis of a flo/lfy homologue isolated from cauliflower (Brassica oleracea L. var. botrytis).

A cDNA library has been constructed from cauliflower curd in which floral development had been initiated. Two cDNAs (pBOFH3 and pBOFH8) have been isolated using the Antirrhinum flo gene as a heterologous probe. The two clones were sequenced and found to contain introns. Comparison of the deduced cDNA sequence of bofh with flo and the Arabidopsis homologue lfy reveals extensive homology. An mRNA transcript of 1.6 kb appears on northern RNA blots. This transcript can be detected, at low levels, before any obvious signs of floral differentiation, reflecting the role bofh plays in determining floral meristem identity.