Identification of common factors regulating suspected gene battery PRESENT: AMIR RUBINSTEIN LIA MITTELMAN 21.01.02

Cancer throughout the animal kingdom Tumors or neoplasms occur throughout the animal kingdom,from the most primitive matazoa to vertebrates. The risk of cancer might be an unavoidable price to pay for the evolution of multicellularity. A fine balance between neoplasms cell proliferation + cell death (cancer)

Genes and cancer 2. tumor suppressors Cancer as a genetic disease, may arise from defects in positive and negative regulators of cell proliferation: 1. oncogenes 2. tumor suppressors

Tumor suppressors and oncogenes

Brain tumor suppresors in drosophila 1.brat 2.mbt 3.lgl 4.dlg Mutants neuroblasts fail to differentiate cotinue to proliferate brain tumor

A common pathway? Common binding sites for transcription factors (“promoter”)? Dlg, Lgl, Mbt, Brat identical phenotype Pathway not yet known.

Regulation of Transcription in Eukaryotes The genes of eukaryotes tend to be larger: Regulatory sequences on the DNA can be located tens of thousands of base pairs away from the transcription start site . A battery of regulatory factors is sometimes needed for proper regulation of certain genes.

Regulation of Transcription in eukaryotes relies on two general classes of cis-acting sequence elements 1. Core promoters

2. Enhancers / Silencers Elements in DNA that are recognized by TRANSCRIPTION FACTORS Enhancers can be located Kbp away from the core promoter! Enhancers are orientation- and position-independent, they may be upstream, downstream, or within a gene.  

Observations about “promoter” elements   1. Individual elements may be present in many copies. Only the TATA box is limited to one.   2. No element is essential ! 3. Only the TATA box displays a conserved position relative to the transcriptional start site.   Other sequences are orientation and position independent. 4.  Transcription factors further regulate levels of expression.

Databases FlyBase Database of genetic and molecular data on all species from the family Drosophilidae. The primary species represented is Drosophila melanogaster.

…Databases Transfac Transcription factor database. Searches for potential transcription factor binding sites. DNA sequence regulatory seq. in DNA TRANSFAC

Analysis - Part 1 We got 1000 bp upstream of each of our 4 genes. -1000

Analysis - Part 2 Brat lgl 4 DNA sequences mbt dlg regulatory seq. in DNA How will similarity look in the results ? We needed something to compare with. Brat lgl mbt dlg TRANSFAC

“Getting started with something we know” Heat-Shock genes Hsp22 hsp27 hsp68 3 genes that are known to have a role in the same biochemical pathway (“gene battery”). They are expressed in stress caused by extreme temperatures. It is known that these genes have at least 1 common DNA binding site for transcription factor, and its sequence is already known.

So this is similarity! Heat-shock genes family

Analysis - Part 3 Now we are ready to check our genes. Here are the results...

Tumor suppressor genes in Drosophila

Explanations Elements of uncertainty in our results: Regulatory sequences appear also downstream (to the 3’ direction after the gene), inside the gene (where introns are found), and further upstream The context in which the short promoter is located within, effects a lot its functionality. So does the 3D structure of the DNA in that area, and the electric distribution of atoms. Far away areas of DNA may fold and effect other areas.

…Explanations Regulatory areas are rich of A’s and T’s. The probability to find a short sequence like AAAT is higher than usual. Some of the “promoters” we found looked like that. Arithmetical manipulation on the results was too simple. We can blame the database. We don’t know whether our 4 genes really act in a common pathway.

Goals for the future… 1. REPORTER GENE TECHNIQUES CAN BE USED TO ISOLATE INDIVIDUAL REGULATORY ELEMENTS OF GENES.

Goals for the future… 2. MICROARRAYS-DNA CHIPS

Thank you for listening !