Gene Expression?–What is a Gene?
A gene codes for a homogeneous ‘functional unit’ – classically a protein, or rRNA, tRNA (other RNAs are emerging). The DNA sequence defines the gene, along with processing steps that determine the product. Generally gene product is a protein which is synthesised by a process of transcription i.e. DNA to mRNA and translation i.e. mRNA to Protein synthesis.
In a given organism every cell has the same genomic DNA, the different characteristics of tissues are the result of differential expression. Which genes are expressed at what stage ? What is the relationship between external or internal signal perception ? All this has important role in gene expression systems. Once activated it is important to know how much of each of those gene products are synthesised. Genome interacts with environment and final products are reproducible, and represent diagnostic expression differences between individuals, between healthy and sick or stressed tissues and as the result of many environmental interactions. What is the function of a gene (or set of genes) in a cell and how does this affect the total biology of the cell (tissue,organism, etc.)
The 3 major strategies for determining the sequence of a gene which could be expressed.
(1) Reverse engineer from the protein, ie sequence the protein and convert the amino acids to appropriate nucleotides. - There is ambiguity because of the redundancy of the genetic code-more than one nucleotide triplet can give the same amino acid. Only when a full-length (m)RNA or protein has been sequenced is there definitive evidence for the existence of a particular gene.
Types of Gene Expression Data Measurement
1. Tag- id (sequence part and record # occurrences)
ESTs, SAGE and MAGE, andMPSS (LYNX).
2. Chromatographic (separation on physical characteristics eg. length, mass, enzyme recognition site).
3. Sequence recognition/amplification (e.g. PCR and internal sequence recognition)
RT-PCR and TaqMan, Beacons
4. Array and chip (use a “known” sequence and intensity)
Nylon, glass and liquidarrays of cDNAs/ESTs and
silicon “chips” ofoligomers (also Northern blots).
The Gene Expression Analysis in the Genomic era depends on :
• High throughput, parallel assays have been developed, in which many genes or gene products are studied simultaneously. This involves:
DNA:High throughput shotgun sequencing or sequencing by hybridization chips
RNA:Microarrays and GeneChips, SAGE, cDNA-AFLP (Functional Genomics,Trancriptomics)
Proteins:Protein arrays, yeast 2-hybrids, MALDI-TOF MSMS (Proteomics, Expressionmonitoring)
Gene silencing: (a) Silencing of transgenes
Transgene silencing falls in the field of epigenetics:
Epigenetics is the study of mitotically/ meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence.
Modes of silencing:
(a) Transcriptional silencing (TGS)i.e. silending of gene and inhibition of mRNA synthesis . It can be of two types: a. cis TGS b. trans TGS
(b) Post-transcriptional silencing (PTGS): Here post transcriptional regulation is carried out between mRNA and protein synthesis: It can also be of two types: a. cis PTGS b. trans PTGS
Post-Transcriptional Gene Silencing (PTGS) was discovered by R. Jorgensen in (1990) in plants. When Jorgensen introduced a re-engineered gene into Petunia that had a lot of homology with an endogenous Petunia gene, both genes became suppressed. This is also called as – Co-suppression. This – suppression was mostly due to increased degradation of the mRNAs (from the endogenous and introduced genes). The regulation was to manipulate pigment synthesis genes in petunia. Genes produced proteins which were enzymes of the flavonoid/ anthocyanin pathway: CHS:chalcone synthase; DFR:dihydroflavonol reductase
Introduction of these genes in Petunia using a strong viral promoter, resulted in drop of mRNA levels and so did pigment levels in many transgenics i.e. genetically modified plants.
RNAi interference is further advanced technique discovered recently
The discovery of this technique resulted in was discovered by Dr. Craig Mello and Dr. Andrew Fire who were awarded 2006 Nobel Prize in Physiology& Medicine, ).
C. elegans (first animal : a nematode) where antisense RNA in vivo was employed.
Control “sense” RNAs also produced suppression of target gene!
Sense RNAs were contaminated with dsRNA and it came out that dsRNA was the suppressing agent. e.g. – Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the nematode C. elegans.
– Antisense RNA or dsRNA for the mex-3 (mRNA) was injected into C. elegans ovaries, and then mex-3 mRNA was detected in embryos by in situ hybridization with a mex-3 probe.
It was concluded that
(1) dsRNA reduced mex-3 mRNA better than antisense mRNA.
(2) the suppressing signal moved from cell to cell.
Mechanism of RNAi: Role of Dicer
1. Cells (plants and animals) undergoing RNAi contained small fragments (~25 nt) of the RNA being suppressed.
2. A nuclease (Dicer) was purified from Drosophila embryos that still had small RNA fragments associated with it, both sense and antisense.
3. The Dicer gene is found in all organisms that exhibit RNAi, and mutating it inhibits the RNAi effect.
Conclusion: Dicer is the endonuclease that degrades dsRNA into 21-24 nt fragments, and in higher eukaryotes also pulls the strands apart via intrinsic helicase activity.