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Large-scale analysis of gene expression: methods and application to the kidney.

Characterization of tissue-specific gene expression profiles, or transcriptomes, may serve two purposes: a) establishing relationships between cell transcriptomes and functions (i.e. molecular and physiological phenotypes) under physiological and pathophysiological conditions serves to elucidate gene functions, and b) determination of the totality of genes expressed in a cell seems a prerequisite for understanding cell functions, because the properties of proteins vary with their environment. Sophisticated methods are now available for transcriptome analysis. They are based on serial, partial sequencing of cDNAs (sequencing of expressed sequenced tags (ESTs) and serial analysis of gene expression (SAGE)), or on parallel hybridization of labeled cDNAs to specific probes immobilized on a grid (macro- and microarrays and DNA chips). Some methods were designed specifically to compare gene expression under different conditions (substractive hybridization, glass microarrays). However, all these methods require several microg of mRNA as starting material, making impossible, in most tissues, to analyse gene expression in homogeneous cell populations. To get around this limitation, we developed a scaled-down SAGE method (SAGE adaptation to downsized extracts: SADE) in our laboratory. SAGE is based on the following: a) each cDNA is characterized by a 10-bp informative sequence called tag, b) the information from several transcripts is condensed into a single DNA molecule by concatenation of several tags, c) sequencing of individual clones from the library of concatemers, computer analysis of sequences and interrogation of sequence databases allow quantitative gene expression profiling. Applied to microdissected mouse nephron segments, SADE made it possible to determine segment-specific transcriptomes.

Animals↗
PubMed1985

Diagnostic library support system for medical practice.

Here is a tragic case where the diagnosis was missed on three separate occasions over a 19-month period. Both terminal conditions are treatable and potentially separable if discovered and aggressively managed. It is not the intent of this presentation to criticize the evaluation of this patient, but rather to point out the need and, also, the potential benefits of an alternate approach, which could have significantly changed the course of this particular patient's evaluation. We have conducted similar analyses with other CPC cases with almost equivalent specificity. These same evaluations have been performed in a real clinical setting with live patient data. What we have learned so far we think will be extremely helpful in extending the potential application of this technology: A text-based decision support tool is only as good as its practitioner. It takes practice and training to learn to use this system effectively. There are many traps in logic, and the use of words and terms within the text must be understood to effectively utilize this tool. There are shortcuts in logical analysis which we mentally use all the time but which cannot be accepted using this system (e.g., you must not rely on your memory or any specific associations to circumvent the system). The system will only become clinically relevant when the entire field of medicine is included in the data base. This is one of our current limitations with only two-thirds of the Merck medical text available for reference. This makes it difficult to apply to a general medical problem since we are not sure which direction the case might take, and often these are multisystem diseases or problems that put us at a severe disadvantage if we don't have the necessary data base. The structure and integrity of the data base are critical to the success of the system. Since numbers are ubiquitous, these cannot be used for key word elements. Techniques must be introduced to create word-oriented numbers that can be uniquely identified (e.g., "AGE14-20"). The publisher of the data must supply a continuous flow of up-to-date material that can be incorporated within the framework of the working system. It is possible to train medical technicians to use the system if they are familiar with medical terminology. The speed and, perhaps, the precision of their analysis could not be expected to rival that of a medical specialist.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1994

A 6.5-Mb yeast artificial chromosome contig incorporating 33 DNA markers on the human X chromosome at Xq22.

The Xq22 region of the human X chromosome contains genes for a number of inherited disorders. Sixty-nine yeast artificial chromosome clones have been isolated and assembled into a 6.5-Mb contig that contains 33 DNA markers localized to this region. This contig extends distally from DXS366 to beyond DXS87 and includes the genes involved in X-linked agammaglobulinemia (btk), Fabry disease (GLA), and Pelizaeus-Merzbacher disease (PLP). The order of markers in this contig is consistent with the known genetic and physical mapping information of Xq22. This cloned material provides a source from which to isolate other genes located in this part of the X chromosome.

Adult↗
PubMed1999

Co-localization of mitochondrial and double minute DNA in the nuclei of HL-60 cells but not normal cells.

In an attempt to isolate genes located on double minute (Dmin) DNA in HL-60 cells, we prepared DNA probe from purified micronuclei. Micronucleation was induced in HL-60 cells by treatment with hydroxyurea. Screening of a cDNA library unexpectedly produced a number of clones containing mitochondrial DNA (mtDNA) sequences. Here, we show that amplified mtDNA sequences were localized in nuclei and micronuclei of HL-60 and COLO 320DM cells, but not in nuclei of WI-38 normal human fibroblasts or peripheral blood T-cells. To unequivocally demonstrate the presence of mtDNA inside of nuclei and micronuclei, we obtained tomographic fluorescence in situ hybridization (FISH) images of mtDNA by confocal microscopy of consecutive sections of paraformaldehyde (PFA)-fixed material. We also located mtDNA in nuclear buds and purified micronuclei. Dmin DNA and mtDNA were always located at similar sites. The mechanisms of nuclear retention of mtDNA and Dmin DNA and the resulting influence on tumorigenesis are discussed.

Cell Cycle↗
PubMed2003

Systematic toxicological analysis: computer-assisted identification of poisons in biological materials.

A new software was developed to improve the chances for identification of a "general unknown" in complex biological materials. To achieve this goal, the total ion current chromatogram was simplified by filtering the acquired mass spectra via an automated subtraction procedure, which removed mass spectra originating from the sample matrix, as well as interfering substances from the extraction procedure. It could be shown that this tool emphasizes mass spectra of exceptional compounds, and therefore provides the forensic toxicologist with further evidence-even in cases where mass spectral data of the unknown compound are not available in "standard" spectral libraries.

Body Fluids↗
PubMed2006

A new strategy for preparing molecular imaging and therapy agents using fluorine-rich (fluorous) soluble supports.

A convenient new strategy for producing radiolabeled compounds in high effective specific activity was developed using soluble fluorous supports. The reported methodology involves a fluorous linker group that is released from the substrate of interest upon reaction with radioiodine. The desired product can then be selectively separated from unreacted starting material and reaction byproducts using a simple fluorous solid-phase extraction procedure. The utility of this approach was demonstrated by labeling a series of benzoic acid derivatives which are commonly used to prepare molecular imaging agents. All compounds were produced in high radiochemical yields, purities, and effective specific activities. The strategy was further elaborated in that it was used to prepare a small collection of radiolabeled benzamides as a way of demonstrating the potential utility of this method for creating libraries of molecular imaging agents.

Benzamides↗
PubMed1991

Alu-primed polymerase chain reaction for regional assignment of 110 yeast artificial chromosome clones from the human X chromosome: identification of clones associated with a disease locus.

Over 400 yeast artificial chromosome (YAC) clones were isolated from the human X chromosome, and 110 of these were assigned to regions defined by chromosome translocation and deletion breakpoints. Polymerase chain reaction using Alu primers was applied to YAC clones in order to generate probes, to identify overlapping clones, and to derive "fingerprints" and sequence data directly from total yeast DNA. Several clones were identified in regions of medical interest. One set of three overlapping clones was found to cross a chromosomal translocation implicated in Lowe syndrome. The regional assignment of groups of YAC clones provides initiation points for further attempts to develop large cloned contiguous sequences, as well as material for investigation of regions involved in genetic diseases.

Base Sequence↗
PubMed1995

Hand-held digital books in radiology: convenient access to information.

Radiologists need constant, convenient access to current information throughout the course of their daily work. Today most learning in radiology is obtained from the printed word in books, journals, and teaching files, supplemented by the spoken word in lectures and conferences. Although learning from printed material and lectures has been proved efficacious over time, these media share the disadvantage of not being conveniently available for reference during the course of daily work at the alternator or in the examination room when accurate and up-to-date information is needed the most. As a result, many important questions about patient care go unanswered. We have developed a technique--hand-held digital books--to lower this barrier to searching and retrieval. When radiologists have a digital library that can be carried with them, they will be able to incorporate current radiology information into their daily decision making. We describe a technique for creating hand-held digital books and their future use in radiology.

Humans↗
PubMed1996

Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties.

We have isolated from a rainbow trout (Oncorhynchus mykiss) spleen cDNA library a clone coding for vimentin. The deduced amino acid sequence reveals a high degree of identity with vimentin from carp (81%), frog (71%), chick and human (73% each). Large stretches in the central alpha-helical rod are identical within all four classes of vertebrates, but in 17 residues spread over the entire rod, the two fish differ distinctly from the tetrapod species. In addition, in the more diverged non-helical head domain, a nonapeptide motif previously shown to be important for regular filament formation is conserved. Recombinant trout vimentin assembles into bona fide filaments in vitro, with a temperature optimum between 18 and 24 degrees C. Above 27 degrees C, however, filament assembly is abruptly abolished and short filaments with thickened ends as well as structures without typical intermediate filament appearance are formed. This distinguishes its assembly properties significantly from amphibian, avian and mammalian vimentin. Also in vivo, after cDNA transfection into vimentin-free mammalian epithelial cells, trout vimentin does not form typical intermediate filament arrays at 37 degrees C. At 28 degrees C, and even more pronounced at 22 degrees C, the vimentin-positive material in the transfected cells is reorganized in the perinuclear region with a partial fibrillar appearance, but typical intermediate filament arrays are not formed. Together with immunoblotting and immunolocalization data from trout tissues, where vimentin is predominantly found in glial and white blood cells, we conclude that vimentin is indeed important in its filamentous form in fish and other vertebrates, possibly fulfilling cellular functions not directly evident in gene targeting experiments carried out in mice.

Amino Acid Sequence↗
PubMed1993

Thymosin beta-10 expression in melanoma cell lines and melanocytic lesions: a new progression marker for human cutaneous melanoma.

When screening a subtraction library for sequences that were specifically expressed in highly metastatic human melanoma cell lines, a cDNA clone was isolated encoding thymosin beta-10. We found that expression of thymosin beta-10 mRNA was associated with metastatic behavior of various human melanoma cell lines in nude mice. Furthermore, Northern blot analysis showed that also in freshly harvested human melanocytic lesions thymosin beta-10 was differentially expressed. Although expression of thymosin beta-10 was also examined in other non-melanoma model systems and materials, no clear relation could be established with metastatic potential or malignancy. Therefore, we conclude that thymosin beta-10 can be considered as a new progression marker for human cutaneous melanoma.

Amino Acid Sequence↗
PubMed1995

Gas phase infrared spectra of 209 polychlorinated biphenyl congeners using gas chromatography with Fourier transform infrared detection: internal standardization with a 13C-labelled congener.

The gas phase infrared spectra of biphenyl and 209 polychlorinated biphenyls (PCB) are observed; 30 are presented in graphical form so that their uniqueness can be appreciated. The spectra were observed with a gas chromatograph linked via a light pipe with a Fourier transform infrared spectrometer. Unequivocal library matches (with match factors > 990 in 1000) can be performed with 50 ng of a congener injected into the chromatograph. Quantitation with the technique was briefly investigated with 2,2',5,5'-tetrachlorobiphenyl (BZ no. 52). Selective wavelength chromatograms of the most intense absorption (1457cm-1) were integrated. Septuplicate injections showed that 20 ng of the compound could be determined with +/- 4% precision. The resolution of the spectrometer was sufficient to distinguish between the 13C-labelled trichlorobiphenyl and the unlabelled material, indicating the possibility of internal or surrogate standardization of PCB analysis.

Carbon Isotopes↗
PubMed2004

Identification of sporulated and vegetative bacteria using statistical analysis of fourier transform mid-infrared transmission data.

A combined mid-infrared spectroscopic/statistical modeling approach for the discrimination and identification, at the strain level, of both sporulated and vegetative bacterial samples is presented. Transmission mode spectra of bacteria dried on ZnSe windows were collected using a Fourier transform mid-infrared (FT-IR) spectrometer. Five Bacillus bacterial strains (B. atrophaeus 49337, B. globigii Dugway, B. thuringiensis spp. kurstaki 35866, B. subtilis 49760, and B. subtilis 6051) were used to construct a reference spectral library and to parameterize a four-step statistical model for the systematic identification of bacteria. The statistical methods used in this initial feasibility study included principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations. Internal cross-validation studies successfully classified 100% of the samples into their correct physiological state (sporulated or vegetative) and identified 67% of the samples correctly as to their bacterial strain. Analysis of thirteen blind samples, which included reference and other bacteria, nonbiological materials, and mixtures of both nonbiological and bacterial samples, yielded comparable accuracy. The primary advantage of this approach is the accurate identification of unknown bacteria, including spores, in a matter of minutes.

Bacillus↗
PubMed2005

Molecular characterization of a consistent 4.5-megabase deletion at 4q28 in prostate cancer cells.

Spectral karyotyping of prostate cell lines LNCaP, DU145, PC3, and 22RV demonstrated structural chromosome rearrangements involving the distal long arm of chromosome 4. In all but 22RV, these are nonreciprocal translocations between chromosomes 4 and 10. In 22RV, an apparently reciprocal t(2q;4q) is seen. Fluorescence in situ hybridization analysis of the chromosome 4 translocation breakpoints demonstrated that deletions were associated with all of the translocations, resulting in a net loss of chromosome material. Overlapping deletions in 4q28 approximately 34 were seen in LNCap, DU145, and 22RV, which defined an approximately 4.5-megabase pair common region of deletion. The deletion in PC3 was more proximal on 4q, involving the 4q21 approximately q26 region. A meta analysis of high-resolution definition of losses of chromosome material from published studies demonstrates that loss of 4q material may occur in at least 50% of primary tumors. This analysis defines a series of genes in the critical 4q region, which is potentially associated with prostate tumor development.

Chromosome Deletion↗
PubMed2006

Rapid analysis of controlled substances using desorption electrospray ionization mass spectrometry.

The recently developed technique of desorption electrospray ionization (DESI) has been applied to the rapid analysis of controlled substances. Experiments have been performed using a commercial ThermoFinnigan LCQ Advantage MAX ion-trap mass spectrometer with limited modifications. Results from the ambient sampling of licit and illicit tablets demonstrate the ability of the DESI technique to detect the main active ingredient(s) or controlled substance(s), even in the presence of other higher-concentration components. Full-scan mass spectrometry data provide preliminary identification by molecular weight determination, while rapid analysis using the tandem mass spectrometry (MS/MS) mode provides fragmentation data which, when compared to the laboratory-generated ESI-MS/MS spectral library, provide structural information and final identification of the active ingredient(s). The consecutive analysis of tablets containing different active components indicates there is no cross-contamination or interference from tablet to tablet, demonstrating the reliability of the DESI technique for rapid sampling (one tablet/min or better). Active ingredients have been detected for tablets in which the active component represents less than 1% of the total tablet weight, demonstrating the sensitivity of the technique. The real-time sampling of cannabis plant material is also presented.

Acetaminophen↗
PubMed2006

Interrogation of genomes by molecular copy-number counting (MCC).

Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the breakpoint without recourse to genomic libraries. The analysis also revealed additional cryptic chromosomal changes close to the translocation junction. MCC is a fast and flexible method for characterizing a wide range of chromosomal aberrations.

Carcinoma, Renal Cell↗
PubMed1991

Expression of endothelin-2 (ET-2) gene in a human renal adenocarcinoma cell line: purification and cDNA cloning of ET-2.

It has been found that human renal adenocarcinoma ACHN cells synthesize and secrete immunoreactive endothelin (ir-ET) in the culture medium. Partial characterization of this material with reverse-phase high-performance liquid chromatography (RP-HPLC) suggested that ACHN cells synthesized only human endothelin-2 (ET-2). Isolation and characterization of this ir-ET-2 has revealed that this peptide has almost the same amino acid sequence and molecular weight as that of human ET-2 deduced from the nucleotide sequence of cloned human ET-2 gene. To delineate the precise structure of human ET-2 precursor, ET-2 cDNAs were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET-2 cDNA that has the longest open reading frame encodes prepro-ET-2 protein, consisting of 178 amino acid residues. The ET-like sequence found in the prepro-ET-1 and -ET-3 was also conserved in this prepro-ET-2. The Northern blot analysis of mRNA revealed that the transcript of the human ET-2 gene was 1.4 kb.

Adenocarcinoma↗
PubMed2002

DNA array profiling of gene expression changes during maize embryo development.

We are using DNA microarray-based gene expression profiling to classify temporal patterns of gene expression during the development of maize embryos, to understand mRNA-level control of embryogenesis and to dissect metabolic pathways and their interactions in the maize embryo. Genes involved in carbohydrate, fatty acid, and amino acid metabolism, the tricarboxylic acid (TCA) cycle, glycolysis, the pentose phosphate pathway, embryogenesis, membrane transport, signal transduction, cofactor biosynthesis, photosynthesis, oxidative phosphorylation and electron transfer, as well as 600 random complementary DNA (cDNA) clones from maize embryos, were arrayed on glass slides. DNA arrays were hybridized with fluorescent dye-labeled cDNA probes synthesized from kernel and embryo poly(A)(+)RNA from different stages of maize seed development. Several characteristic developmental patterns of expression were identified and correlated with gene function. Patterns of coordinated gene expression in the TCA cycle and glycolysis were analyzed in detail. The steady state level of poly(A)(+) RNA for many genes varies dramatically during maize embryo development. Expression patterns of genes coding for enzymes of fatty acid biosynthesis and glycolysis are coordinately regulated during development. Genes of unknown function may by assigned a hypothetical role based on their patterns of expression resembling well characterized genes. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s10142-002-0046-6.

Blotting, Northern↗
PubMed1994

Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones.

High-throughput automated partial sequencing of anonymous cDNA clones provides a method to survey the repertoire of expressed genes from an organism. Comparison of the coding capacity of these expressed sequence tags (ESTs) with the sequences in the public data bases results in assignment of putative function to a significant proportion of the ESTs. Thus, the more than 13,400 plant ESTs that are currently available provide a new resource that will facilitate progress in many areas of plant biology. These opportunities are illustrated by a description of the results obtained from analysis of 1500 Arabidopsis ESTs from a cDNA library prepared from equal portions of poly(A+) mRNA from etiolated seedlings, roots, leaves, and flowering inflorescences. More than 900 different sequences were represented, 32% of which showed significant nucleotide or deduced amino acid sequences similarity to previously characterized genes or proteins from a wide range of organisms. At least 165 of the clones had significant deduced amino acid sequence homology to proteins or gene products that have not been previously characterized from higher plants. A summary of methods for accessing the information and materials generated by the Arabidopsis cDNA sequencing project is provided.

Amino Acid Sequence↗