Full sequences have been developed for a number of organisms including human, yeast, Drosophila and others.
Positional cloning is done to identify an unknown gene associated with a trait of interest. The gene is mapped as closely as possible to existing markers. Sequencing is performed in the area of the markers to identify possible genes associated with the trait. This may involve sequencing of multiple BACs in a given region. "Chromosome walking" involves moving along a chromosome region until the gene of interest is identified. Information about gene orders in related species may be useful for identifying the gene if gene order is similar in the other species.
Analysis of the sequence of cDNAs from a given tissue ("expressed sequence tages, or ESTs) can provide insights into gene expression patterns in tissues. Some tissues may have more expressed genes than others. The abundance of specific sequences can give a measure of levels of expression of specific genes.
Regulation of gene expression is a central problem in molecular genetics. Some common principles and differences are evident between prokaryotes and eukaryotes in this area. Five major control points have been identified for controlling gene expression. (1) DNA may be altered, rearranged or modified. (2) Transcription may be controlled, primarily by the binding of proteins to DNA. (3) Processing of RNA may be regulated. (4) The stability of mRNA may vary . (5) Translation may be controlled.
Prokaryotes and eukaryotes have differences with respect to regulating gene expression. Prokaryotes are single-celled, eukaryotes are often multicellular. Prokaryotes have few genes, eukaryotes have many. Prokaryotes have little extra DNA, eukaryotes have a lot. Prokaryotes need to regulate genes primarily to be able to respond to environmental change, eukaryotes need to regulate genes primarily to allow tissue-specific expression and for control of genes in development.
Gene expression is described by certain terms. Negative regulation of transcription means that protein binding shuts off transcription. This is most common in prokaryotes. Positive regulation of transcription means that protein binding facilitates transcription. This is most common in eukaryotes. Inducible means that the presence of a small molecular facilitates gene expression. (Ex: lactose in Lac operon). Generally common in pathways involved in breaking down energy sources. Repressible means that presence of a small molecule shuts off transcription. (Ex: trp in trp operon). This is generally common in pathways involved in synthesizing cell components.
Prokaryotes were studied in detail before eukaryotes were studied. The Lac Operon is a classic system for studying control of gene expression. E. coli need to regulate the enzymes B-galactosidase (Z gene product, which cleaves lactose into glucose + galactose) and permease (Y gene product, which helps lactose enter the cell) which are involved in utilizing lactose sugar. It is most efficient to synthesize these enzymes when lactose is present and not otherwise. The system was initially worked out using isolation of mutants, genetic mappng with transduction, and the construction and study of partial diploids carrying an extra Lac operon on the F factor (F’). The wild type Lac operon results in transcription of the Z and Y genes when lactose is present and not when it is absent. This is accomplished by having a repressor (I gene product) bind to the operator (O site, near the promoter) when lactose is absent, shutting off transcription. When lactose is present, it binds the repressor which then can no longer bind to the O site. This allows transcription of the genes. Various mutants were used in working out the system. Z — mutants cannot produce B-galactosidase enzyme. Oc mutants have operators which cannot bind the repressor. Their phenotype is constant expression of the Z and Y genes.