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Genomic Studies of Renal Cells in Space

Dr. Timothy Hammond, Tulane University

New Technology: Automated Gene Displays

The human genome project, and the associated production of huge libraries of expressed sequence tags (ESTs), provided momentum for the development of new methods to assay large numbers of genes, gene mutations and single point mutations simultaneously. Gene (expression) array analysis has provided a rapid, inexpensive, but sophisticated method to meet these needs. Gene array is a newly emerging technology. It has only been two and a half years since the first gene arrays of a few hundred genes were made at Patrick Brown's lab at Stanford University. Now there are arrays containing tens of thousands of genes, and it is estimated that by 2005, the entire human genome will be available on a single array. There has been much academic and press speculation that gene array will revolutionize the search for new drugs by monitoring the drug's effects on thousands of genes, making it possible to predict their usefulness as therapeutic agents. Today there are two multi-million dollar companies [Synteni/Incyte and Affymetrix] and a few small startup companies supplying this technology to the pharmaceutical industry. There are currently around ten research labs in the country that have the ability to perform gene array analysis.

Gene expression assay using a micro array can be summarized as follows. Each cDNA to be assayed is identified, and isolated. Either sequence specific oligos are manufactured, or PCR is used to generate countless copies of the individual clones. Approximately 10,000 copies of the first clone are spotted with a robot in the center of the first square of an array. If the array were a chessboard, there would be 64 squares with a different unique clone spotted in a circle in the middle of each square. In our array, up to 10,000 individual clones can be spotted on a 1-cm square on the surface of a microscope slide. The sample(s) to be assayed for gene expression are then prepared by multiplex PCR of genomic DNA. This technique employs multiple sets of PCR primers, designed with similar annealing temperatures, in a single reaction tube. Temperature-dependence of the PCR elongation reaction potently prevents nesting of the primers, or generation of longer PCR products between unpaired primer sets. PCR techniques are used to incorporate a green (cyanine-3) or red (cyanine-5) dye into the multiplex PCR products. An unknown sample can then be compared to a control sample by competitive annealing on the array, or a single sample annealed. The resultant fluorescent array is read quantitatively by a laser based fluorescent scanner.

[Example I. Gene array analysis derived from human renal cortical cells flown on STS-90 "Neurolab" and ground based control cultures.]

Many cell cultures flown in microgravity undergo dramatic changes in the fidelity of their differentiated structural and biochemical features. To test our hypothesis that tissue differentiation is determined by 3-dimentionality, levels of turbulence and shear, and cospatial relationships of cells of dissimilar sedimentation and size, we flew human renal cells on space shuttle mission STS-90 ìNeurolabî and made comparison to ground based controls.

On recovery of flight and ground samples, RNA was extracted and automated gene array analysis of the expression of 10,000 genes performed. To achieve this the polyA RNA from flight and ground where reverse transcribed with fluorescent bases tagged with different colors. A competition binding analysis was performed by annealing the fluorescent probes competitively to 10,000 cDNAs immobilized in a grid on a glass microscope slide. The bound fluorescent DNA was quantitated with a fluorescent reader, and patterns analyzed utilizing GEMTools software.

The findings in summary are:

a. a select but substantial group of more than 800 genes changed up and down in microgravity

b. the genes which changed in microgravity were independent of known shear stress response element dependent genes and heat shock proteins

c. six specific transcription factors underwent large changes in microgravity including the Wilm's tumor zinc finger protein and the vitamin D receptor

Hence, it appears that there is a select group of gravity dependent genes. Candidate transcription factors are being be defined.

For a relevant comparison we analyzed the genes which change in hypergravity (3g) during a centrifuge experiment. In terms of our hypothesis this provided a cell culture modality with no 3-dimentionality, low levels of turbulence and substantial shear, and near perfect cospatial relationships. We predicted that these conditions should be very poorly optimal for renal tissue differentiation and there should be little detectable change in gene expression. Our prediction was largely accurate. The best ground based simulation of conditions in space, the rotating wall vessel showed a pattern of gene expression midway between the flight and centrifuge samples.

[Example II: Yeast : demonstration of gene array on an organim which has been completely cloned.]

Other labs have created a comprehensive catalog of yeast genes whose transcript levels vary in a time dependent manner during meiosis. And in response to changes in culture conditions. The use of DNA microarrays and samples of RNA from yeast cultures to determine the clustering of the groups of genes, which change with division and or injury was demonstrated. Using periodicity and correlation algorithms groups of genes that have similar patterns of change can be identified. Furthermore, analysis of these sets of genes for promoter elements defines the mechanisms mediating these genetic changes.

Timothy Hammond
Tulane Environmental Astrobiology Center
New Orleans LA 70112