Northern blot

Northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot. Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.

A northern blot is very similar to a Southern blot except that it is RNA rather than DNA which is extracted, run on a gel and transferred to a filter membrane. There are 3 types of RNA: tRNA (transfer RNA - active in assembly of polypeptide chains), rRNA (ribosomal RNA - part of the structure of ribosomes) and mRNA (messenger RNA - the product of DNA transcription and used for translation of a gene into a protein). It is mRNA which is isolated and hybridized in northern blots.








* mRNA is extracted from the cells grown in galactose and cells grown in glucodse and purified.
* The mRNA is loaded onto a gel for electrophoresis. Lane 1 has gal mRNa Lane 2 has the Glucose mRNA.
* An electric current is passed through the gel and the RNA moves away from the negative electrode. The distance moved depends on the size of the RNA fragment. Since genes are different sizes the size of the mRNAs varies also. This results in a smear on a gel. Standards are used to quantitate the size. The RNA can be visualized by staining first with a fluorescent dye and then lighting with UV.
* RNA is single-stranded, so it can be transferred out of the gel and onto a membrane without any further treatment. The transfer can be done electrically or by capillary action with a high salt solution.
* A GAL DNA probe is incubated with the blot.the single stranded GAL DNA probe binds with immobilized GAL mRNA The blot is washed to remove non-specifically bount probe and then a development step allows visualization of the probe that is bound.

Control of Gene Expression

This lecture begins by illustrating how cells differ in the proteins theye xpress. The same cell may express different proteins at different times in its life, and in multi-cellular organisms, different types of cells typically express different suites of proteins. Given that all cells in an organism have the same set of genes, how do these differences in protein expression arise? The lecture outlines the history of the original experiments done by Jacques Monod and François Jacob in the late 1950s and early 1960s using bacteria to discover the basic mechanisms of gene regulation.


Differences in cell function often produce different cell shapes, but the critical difference is the different protein mix each type of cell contains.

Though many thousands of proteins are coded for in a typical eukaryotic cell, most cells contain only a fraction of that number at any one time. This is the case even in single-celled bacteria. This situation makes sense because making proteins is expensive and many proteins do not store well. This leads to the question of how cells control protein production.
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We know that making proteins requires the transcription of DNA into mRNA, then the translation of mRNA into proteins; thus, we could alternatively ask why all genes in a cell are not always transcribed and translated.

The general answer is that cells regulate gene expression by controlling the conditions necessary for transcription and translation.


The complexity of gene expression means that there are many possible points where cells can control it.

A. In addition to transcription and translation, the initial mRNA transcript must be processed before it is translated, and the final polypeptide must often be further modified by enzymes before it is functional.
B. Cells could theoretically control gene expression at any of these points,but the majority of gene regulation happens at the level of transcription—specifically, by controlling mRNA synthesis.C. This is the case primarily for efficiency; it “costs” cells less to prevent the process from starting in the first place.


III. Control of transcription mechanisms involves genetic “lock-and-key”mechanisms, which work differently in prokaryotes and eukaryotes.

A. To a first approximation, the “switch” that turns transcription on and off is a lock-and-key mechanism.
B. The lock is a specific sequence of nucleotide bases on the DNA that is distinct from but often located physically adjacent to the gene it controls. Each gene has one or more of these regulatory regions, which normally occur “upstream” from the gene.
C. The key is usually a protein with the right shape (including not only physical shape but the correct physical and chemical properties of amino acids) to fit the DNA lock. In general, only one protein will bind to a particular regulatory region.
D. In order to start transcription, RNA polymerase must bind to promoter sites on the DNA molecule.The binding of RNA polymerase to a promoter is not specific; thus, regulatory regions and proteins generally act in two ways to control gene expression.

1. In prokaryotic cells, regulatory regions typically lie in between promoter sites and genes. Regulatory proteins bound to regulatory regions physically block RNA polymerase from reaching the gene.This represents negative control and is typical of prokaryotic cells.
2. The other type of control depends on the fact that RNA polymerase cannot always bind efficiently to the promoter by itself. In this case regulatory proteins nteracting with regulatory regions affect the ability of RNA polymerase to bind to a promoter. In the most general case, regulatory proteins (called “transcription factors”) facilitate the binding of RNA polymerase, resulting in positive control, though these proteins can have either a positive or negative effect on binding in specific cases.
3. These types of controls create two contrasts: physical blockage of RNA polymerase versus an effect on its ability to bind to a promoter region on the DNA, and negative control versus positive control.

E. Though the lock-and-key mechanis m for gene regulation can be compared to turning on a car, there are some important differences.
1. The “driver” (RNA polymerase) is not selective as to which “car” it chooses. RNA polymerase simply tries to transcribe every gene.
2. Some genes will be exp ressed only if the key is not in the regulatory lock.
3. Some genes require a combination of many different keys to be expressed, and some keys must be in their locks, while others must be out.

IV. In the late 1950s and early 1960s, Jacques Monod and François Jacob investigated how gene regulation works in prokaryotes.
A. The bacteria Escherichia coli (E. coli for short) requires several enzymes to metabolize lactose. Biochemists discovered in the early 20th
century that E. coli produces these enzymes only when lactose is available, suggesting that lactose induces enzyme production.
B. This observation led Jacques Monod and François Jacob to the question of how lactose could induce gene expression; they found mutant forms
of E. coli that differed in how they expressed lactose-digesting enzymes.
1. Two kinds of mutants were unable to digest lactose; in one case, an enzyme involved in lactose breakdown itself was defective, while
in another case, an enzyme that brings lactose into the cell was defective. These mutations fit into the expected pattern: A
mutation in a gene causes a coding error that produces a dysfunctional protein, which cannot do its job.
2. A third kind of mutant always produced lactose-digesting enzymes, regardless of whether lactose was present or not. This type of mutation produced a gain of constant function rather than a loss of function. Monod and Jacob inferred that this third type of mutant must have a mutation in some protein involved in
controlling gene expression, not in the genes coding for the enzymes themselves.
C. Monod and Jacob’s idea was revolutionary at the time, because it pointed to a protein whose sole function was to regulate the expression
of other genes.
D. In E. coli, the regulatory protein seemed to inhibit enzyme production.After some debate, physicist Leo Szilard prevailed with the idea that the regulatory protein acted as a repressor that normally preventedproduction of the lactose-digesting enzymes. Szilard’s model suggested that lactose acts as an inducer by disabling the repressor protein; that is,expression of the lactose-digesting enzymes is under negative control.