An Overview of Gene Expression

Overview

Gene expression is the complex process where a cell uses its genetic information to make

functional products. This process is regulated at multiple stages, and any misregulation could lead

to diseases such as cancer.

This video highlights important historical discoveries relating to gene expression, including the

understanding of how distinct combinations of DNA bases encode the amino acids that make up

proteins. Key questions in the field of gene expression research are explored, followed by a

discussion of several techniques used to measure gene expression and investigate its regulation.

Finally, we look at how scientists are currently using these techniques to study gene expression.

Procedure
Log in or Start trial to access full content. Learn more about your institution’s access to JoVE

content here

Gene expression is the process where information contained in a cell’s DNA is used to

make functional products. This carefully orchestrated procedure is regulated at several

stages, and misregulation can often result in diseases such as cancer.

This video provides an overview of the history of gene expression research, key

questions, methods in the field, and how these techniques are being applied.

We’ll begin by reviewing some major discoveries about gene expression.

The first convincing model for how DNA might carry genetic information was

established in 1953, when Francis Crick and James Watson, with help from Rosalind

Franklin’s data, solved the structure of DNA—a double helix made of two linear chains

of nucleotide bases that are arranged in a defined, but infinitely variable, sequence.
Five years later, Crick proposed two important ideas that would form the backbone of

our understanding of gene expression. His “sequence hypothesis” suggested that

DNA’s nucleotide sequence is used, via an unstable RNA intermediate, as a code for

proteins’ amino acid sequences. At the same time, his “central dogma” hypothesized

the different flows of genetic information that can occur, and in particular, held that

information cannot be transferred from protein back to nucleic acids.

In 1960, François Jacob and Jacques Monod—through their work on lactose-

metabolizing genes in bacteria—proposed a model for the regulation of gene

expression. They suggested that the expression of “structural genes,” which perform

structural or enzymatic functions, is controlled by the products of “regulatory genes”

that bind to adjacent regulatory sites. We now know that substantially similar modes of

gene regulation, mediated by proteins called transcription factors, occur in all

organisms.

A year later, in 1961, Jacob—along with Sydney Brenner—discovered messenger or “m”-

RNA as the unstable intermediate between DNA and proteins proposed by Crick. That

same year, Brenner and Crick began to crack the “genetic code,” which dictates how
information in DNA encodes proteins. They determined that each triplet of adjacent

nucleotides, or a “codon,” specifies one of the 20 amino acids that constitute proteins.

Over the next few years, researchers led by Marshall Nirenberg, Har Gobind Khorana,

and Severo Ochoa used multiple approaches to define the amino acids encoded by all

64 possible codons. With the cracking of the genetic code, scientists continued to

investigate how gene expression is regulated.

A major discovery came in 1974, when Roger Kornberg and colleagues showed that

DNA in eukaryotic cells, such as those of animals and plants, is “wrapped” around

complexes of histone proteins, yielding structures now called “nucleosomes.” We now

know that changes in chromatin structure play important roles in gene regulation.
Another twist came in 1977, when Phil Sharp and Rich Roberts found that mRNA

sequences were not entirely complementary to their corresponding DNA templates.

Certain “missing regions,” now called introns, are removed from between the protein-

coding exons in the mature RNA transcript in a process known as “splicing.” Five years

later, the research group of Ronald Evans demonstrated that “alternative” splicing of

the same transcript could produce variants, or “isoforms,” of the same protein with

different functions.

Since the 1990s, our understanding of the complexity of gene regulatory networks

expanded dramatically with the discovery of RNA-mediated gene silencing. We now

know that several different families of small RNAs, with members that are 20−30

nucleotides in size, regulate gene expression in a variety of ways.

After reviewing the history of gene expression research, let’s look at some major

questions in the field.

One topic being investigated is how transcription factors regulate genes. Scientists are

not only interested in identifying the genomic sequences bound by transcription

factors, but are also looking at how regulatory proteins interact with one another to
integrate signals and regulate gene expression.

Other researchers study alternative splicing, and how this process is regulated in

different biological contexts. In addition, some of them are trying to determine whether

protein isoforms always have different, distinct functions.

Finally, many researchers are investigating the mechanism of action of small RNAs,

and are trying to identify their regulatory targets. There is also a growing interest in

whether small RNAs can be used as “biomarkers” to diagnose diseases.

Now, let’s look at the tools researchers use to assess gene expression.
A popular method is reverse transcription, or “RT”-PCR, which converts RNA into

complementary or “c”-DNA before subjecting it to amplification. By including

fluorescent molecules that are incorporated into the DNA during PCR, it’s possible to

use this technique to quantitatively measure gene expression and observe the results in

“real-time.”

To simultaneously assess the expression of thousands of genes, microarrays can be

used. Here, DNA sequences are “printed” on slides, which are then hybridized to

fluorescent probes generated from sample RNA. The resulting pattern of fluorescence

can be used to identify expressed genes.

Another technique to profile gene expression is to sequence the transcriptome, or all of

the expressed RNAs in a cell. Here, cDNA generated from RNA samples is subjected to

high-throughput sequencing. Unlike microarrays, transcriptome sequencing does not

require preexisting genomic information, and can be used to identify unknown

transcripts or novel gene isoforms.

Researchers can also visually evaluate where a gene is expressed using in

situ hybridization. In this technique, RNA is first hybridized with complementary probes,

color or fluorescence signal.

The reporter assay is another technique that can provide insight into gene regulation.

The reporter gene product generates a signal such as color or fluorescence. The

reporter maybe fused directly to a gene of interest, or be placed under the control of a

regulatory sequence, such as the promoter that drives a gene’s transcription, or a more

distant enhancer element. The reporter signal can then act as readout for the regulatory

element’s activity, or the expression pattern of the gene of interest.

sites that transcription factors bind to when regulating gene expression. Here,

complexes of proteins and the DNA they bind are isolated by antibodies, and the target

DNA is identified by PCR or sequencing.

After surveying the methods for studying gene expression, let’s look at some of their

applications.

Cells within a population may exhibit subtle differences in gene expression that can

have biological consequences. In this study, researchers placed individual human

embryonic stem cells from the same culture into separate wells on a plate. Using

quantitative RT-PCR, scientists determined that the expression of Nanog—a stem cell

“marker”—differed within a sample.

Some researchers investigate whether different isoforms of regulatory proteins

function differently. Here, ChIP was applied to human immune cells to identify the

binding targets of a protein’s “long” and “short” isoforms. Sequencing results showed

that some gene targets were only recognized by the short isoform, pointing to potential

functional differences.

Finally, reporter assays can be used to evaluate gene regulation mediated by small

RNAs, such as microRNAs. As microRNAs can inhibit gene expression by binding to the

3¢ untranslated regions of mRNAs, scientists attached this region from different genes

to a luciferase reporter, and introduced each of them into cells along with a microRNA.

Gene targets of the microRNA were then identified by looking for cells with decreased

luminescence signal.

You’ve just watched JoVE’s introduction to gene expression. We’ve reviewed major

findings in gene expression research, prominent questions and methods in the field,

and some current applications.