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Gene Expression: The phenotypic manifestation of a gene or genes by the processes of Genetic transcription and Genetic translation.

An Overview of Gene Expression

JoVE 5546

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…

 Genetics

What is Gene Expression?

JoVE 10797

Gene expression is the process in which DNA directs the synthesis of functional products, such as proteins. Cells can regulate gene expression at various stages. It allows organisms to generate different cell types and enables cells to adapt to internal and external factors.

A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is made up of nucleotides and proteins consist of amino acids, a mediator is required to convert the information that is encoded in DNA into proteins. This mediator is the messenger RNA (mRNA). mRNA copies the blueprint from DNA by a process called transcription. In eukaryotes, transcription takes place in the nucleus by complementary base-pairing with the DNA template. The mRNA is then processed and transported into the cytoplasm where it serves as a template for protein synthesis during translation. In prokaryotes, which lack a nucleus, the processes of transcription and translation occur at the same location and almost simultaneously since the newly-formed mRNA is susceptible to rapid degradation. Every cell of an organism contains the same DNA, and consequently the same set of genes. However, not all genes in a cell are “turned on” or use to synthesize proteins. A gene is said to be “expressed” when the protein it encodes is produced by the cell. Gen

 Core: Biology

Expression Profiling with Microarrays

JoVE 5547

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, …

 Genetics

Gene Silencing with Morpholinos

JoVE 5326

Morpholino-mediated gene silencing is a common technique used to study roles of specific genes during development. Morpholinos inhibit gene expression by hybridizing to complementary mRNAs. Due to their unique chemistry, morpholinos are easy to produce and store, which makes them remarkably cost effective compared to other gene silencing methods.


This video reviews proper…

 Developmental Biology

Genetic Engineering of Model Organisms

JoVE 5327

Transgenesis, or the use of genetic engineering to alter gene expression, is widely used in the field of developmental biology. Scientists use a number of approaches to alter the function of genes to understand their roles in developmental processes. This includes replacement of a gene with a nonfunctional copy, or adding a visualizable tag to a gene that allows the resultant fusion protein to …

 Developmental Biology

An Introduction to Developmental Genetics

JoVE 5325

Development is the complex process through which a single-celled embryo transforms into a multicellular organism. Developmental processes are guided by information encoded in an organism's DNA, and geneticists are trying to understand how this information leads to a fully formed organism.


This video reviews seminal research in the field of developmental biology, including the…

 Developmental Biology

Chromatin Immunoprecipitation

JoVE 5551

Histones are proteins that help organize DNA in eukaryotic nuclei by serving as “scaffolds” around which DNA can be wrapped, forming a complex called “chromatin”. These proteins can be modified through the addition of chemical groups, and these changes affect gene expression. Researchers use a technique called chromatin immunoprecipitation (ChIP) to …

 Genetics

Neuronal Transfection Methods

JoVE 5215

Transfection - the process of transferring genetic material into cells - is a powerful tool for the rapid and efficient manipulation of gene expression in cells. Because this method can be used to silence the expression of specific proteins or to drive the expression of foreign or modified proteins, transfection is an extremely useful tool in the study of the cellular and…

 Neuroscience

Whole-Mount In Situ Hybridization

JoVE 5330

Whole-mount in situ hybridization (WMISH) is a common technique used for visualizing the location of expressed RNAs in embryos. In this process, synthetically produced RNA probes are first complementarily bound, or "hybridized," to the transcripts of target genes. Immunohistochemistry or fluorescence is then used to detect these RNA hybrids, revealing spatial and temporal patterns of…

 Developmental Biology

Organization of Genes

JoVE 10786

The genomes of eukaryotes can be structured in several functional categories. A strand of DNA is comprised of genes and intergenic regions. Genes themselves consist of protein-coding exons and non-coding introns. Introns are excised once the sequence is transcribed to mRNA, leaving only exons to code for proteins.

In eukaryotic genomes, genes are separated by large stretches of DNA that do not code for proteins. However, these intergenic regions carry important elements that regulate gene activity, for instance, the promoter where transcription starts, and enhancers and silencers that fine-tune gene expression. Sometimes these binding sites can be located far away from the associated gene. As researchers investigated the process of gene transcription in eukaryotes, they realized that the final mRNA that codes for a protein is shorter than the DNA it is derived from. This difference in length is due to a process called splicing. Once pre-mRNA has been transcribed from DNA in the nucleus, splicing immediately removes introns and joins exons together. The result is protein-coding mRNA that moves to the cytoplasm and is translated into protein. One of the largest human genes, DMD, is over two million base pairs long. This gene encodes the muscle protein dystrophin. Mutations in DMD cause muscular dystrophy, a disorder characteri

 Core: Biology

An Introduction to Stem Cell Biology

JoVE 5331

Cells that can differentiate into a variety of cell types, known as stem cells, are at the center of one of the most exciting fields of science today. Stem cell biologists are working to understand the basic mechanisms that regulate how these cells function. These researchers are also interested in harnessing the remarkable potential of stem cells to treat human diseases.


Here,…

 Developmental Biology

RNA-Seq

JoVE 5548

Among different methods to evaluate gene expression, the high-throughput sequencing of RNA, or RNA-seq. is particularly attractive, as it can be performed and analyzed without relying on prior available genomic information. During RNA-seq, RNA isolated from samples of interest is used to generate a DNA library, which is then amplified and sequenced. Ultimately, RNA-seq can …

 Genetics

Types of RNA

JoVE 10800

Three main types of RNA are involved in protein synthesis: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). These RNAs perform diverse functions and can be broadly classified as protein-coding or non-coding RNA. Non-coding RNAs play important roles in the regulation of gene expression in response to developmental and environmental changes. Non-coding RNAs in prokaryotes can be manipulated to develop more effective antibacterial drugs for human or animal use. The central dogma of molecular biology states that DNA contains the information that encodes proteins and RNA uses this information to direct protein synthesis. Different types of RNA are involved in protein synthesis. Based on whether or not they encode proteins, RNA is broadly classified as protein-coding or non-coding RNA. Messenger RNA (mRNA) is the protein-coding RNA. It consists of codons—sequences of three nucleotides that encode a specific amino acid. Transfer RNA (tRNA) and ribosomal RNA (rRNA) are non-coding RNA. tRNA acts as an adaptor molecule that reads the mRNA sequence and places amino acids in the correct order in the growing polypeptide chain. rRNA and other proteins make up the ribosome—the seat of protein synthesis in the cell. During translation, ribosomes move along an mRNA strand where they stabilize the binding of tRNA molecules and catalyze the for

 Core: Biology

RNA Analysis of Environmental Samples Using RT-PCR

JoVE 10104

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz


Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies…

 Environmental Microbiology

RNA Splicing

JoVE 10802

The process in which eukaryotic RNA is edited prior to protein translation is called splicing. It removes regions that do not code for proteins and patches the protein-coding regions together. Splicing also allows several protein variants to be expressed from a single gene and plays an essential role in development, tissue differentiation, and adaptation to environmental stress. Errors in splicing can lead to diseases such as cancer. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts designated to become mRNA are called precursor messenger RNA (pre-mRNA). The pre-mRNA is then processed to form mature mRNA that is suitable for protein translation. Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins whereas introns are the non-coding regions. RNA splicing is the process by which introns are removed and exons patched together. Splicing is mediated by the spliceosome—a complex of proteins and RNA called small nuclear ribonucleoproteins (snRNPs). The spliceosome recognizes specific nucleotide sequences at exon/intron boundaries. First, it binds to a GU-containing sequence at the 5’ end of the intron and to a branch point sequence containing an A towards the 3’ end of the intron. In a number of carefully-orches

 Core: Biology

An Introduction to Cellular and Molecular Neuroscience

JoVE 5213

Cellular and molecular neuroscience is one of the newest and fastest growing subdisciplines in neuroscience. By investigating the influences of genes, signaling molecules, and cellular morphology, researchers in this field uncover crucial insights into normal brain development and function, as well as the root causes of many pathological conditions.


 Neuroscience

Antibiotic Selection

JoVE 10807

Researchers use antibiotic resistance genes to identify bacteria that possess a plasmid containing their gene of interest. Antibiotic resistance naturally occurs when a spontaneous DNA mutation creates changes in bacterial genes that eliminate antibiotic activity. Bacteria can share these new resistance genes with their offspring and other bacteria. The overuse and misuse of antibiotics have created a public health crisis, as resistant and multi-resistant bacteria continue to develop. Antibiotics, such as penicillin, are drugs that kill or stop bacterial growth. Bacteria that naturally or artificially acquired antibiotic resistance genes do not respond to antibiotics. Scientists exploit this by designing plasmids—small, self-replicating pieces of DNA—that carry both an antibiotic resistance gene and a gene of interest. Antibiotic resistance is an integral part of DNA cloning that allows a researcher to identify cells that absorbed a DNA of interest. The researcher’s DNA of interest is introduced into bacterial cells using a process called transformation. Bacterial transformation involves temporarily creating small holes in the bacterial cell wall to permit the uptake of external DNA such as a plasmid. Only some bacterial cells absorb new DNA. Since the plasmid includes both the DNA of interest and a gene that confers resistance to a spe

 Core: Biology

An Introduction to Molecular Developmental Biology

JoVE 5328

Molecular signals play a major role in the complex processes occurring during embryonic development. These signals regulate activities such as cell differentiation and migration, which contribute to the formation of specific cell types and structures. The use of molecular approaches allows researchers to investigate these physical and chemical mechanisms in detail.


This video will …

 Developmental Biology

DNA Methylation Analysis

JoVE 5550

Methylation at CpG dinucleotides is a chemical modification of DNA hypothesized to play important roles in regulating gene expression. In particular, the methylation of clusters of methylation sites, called “CpG islands”, near promoters and other gene regulatory elements may contribute to the stable silencing of genes, for example, during epigenetic processes…

 Genetics

Epigenetic Regulation

JoVE 10803

Epigenetic mechanisms play an essential role in healthy development. Conversely, precisely regulated epigenetic mechanisms are disrupted in diseases like cancer.

In most mammals, females have two X chromosomes (XX) while males have an X and a Y chromosome (XY). The X chromosome contains significantly more genes than the Y chromosome. Therefore, to prevent an excess of X chromosome-linked gene expression in females, one of the two X chromosomes is randomly silenced during early development. This process, called X-chromosome inactivation, is regulated by DNA methylation. Scientists have found greater DNA methylation at gene promoter sites on the inactive X chromosome than its active counterpart. DNA methylation prevents the transcription machinery from attaching to the promoter region, thus inhibiting gene transcription. Abnormal DNA methylation plays an important role in cancer. The promoter region of most genes contains stretches of cytosine and guanine nucleotides linked by a phosphate group. These regions are called CpG islands. In healthy cells, CpG islands are not methylated. However, in cancer cells, CpG islands in the promoter regions of tumor suppressor genes or cell cycle regulators are excessively methylated. Methylation turns off the expression of these genes, allowing cancer cells to divide rapidly and uncontrollably.

 Core: Biology

Transgenic Organisms

JoVE 10809

Transgenic organisms are genetically engineered to carry transgenes—genes from a different species—as part of their genome. The transgene may either be a different version of one of the organism’s genes or a gene that does not exist in their genome. Transgenes are usually generated by recombinant DNA and DNA cloning techniques. Transgenic bacteria, plants, and animals allow scientists to address biological queries and design practical solutions. Scientists begin the process of transgenesis—introducing a transgene into an organism’s genome—by selecting an appropriate technique. There are several biological, chemical, and physical methods of transgenesis. A common biological method involves the virus-mediated introduction of foreign DNA into a host cell genome, called transduction. A popular chemical method uses calcium phosphate (Ca3(PO4)2). The method is based on the formation of a Ca3(PO4)2/DNA precipitate to facilitate DNA binding to and entering cells. Physical methods such as microinjection—a technique that uses a thin, glass needle to manually insert genetic material into cells—artificially introduce DNA by force. Once inside the cell, a transgene can either integrate randomly or at a specific site in the genome with the help of DNA repa

 Core: Biology

Explant Culture for Developmental Studies

JoVE 5329

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo approach allows researchers to manipulate and observe developing tissues in ways that are not possible in vivo. Once established, explant culture is frequently used to understand the role of genes and signaling molecules in …

 Developmental Biology

Bacterial Signaling

JoVE 10713

At times, a group of bacteria behaves like a community. To achieve this, they engage in quorum sensing, the perception of higher cell density that results in a shift in gene expression. Quorum sensing involves both extracellular and intracellular signaling. The signaling cascade starts with a molecule called an autoinducer (AI). Individual bacteria produce AIs that move out of the bacterial cell membrane into the extracellular space. AIs can move passively along a concentration gradient out of the cell, or be actively transported across the bacterial membrane. When cell density in the bacterial populations is low, the AIs diffuse away from the bacteria, keeping the environmental concentration of AIs low. As bacteria reproduce and continue to excrete AIs, the concentration of AIs increases, eventually reaching a threshold concentration. This threshold permits AIs to bind membrane receptors on the bacteria, triggering changes in gene expression across the whole bacterial community. Many bacteria are broadly classified as gram positive or gram negative. These terms refer to the color that the bacteria take on when treated with a series of staining solutions which were developed by Hans Christian Joachim Gram over a century ago. If bacteria pick up a purple color, they are gram-positive; if they look red, they are gram-negative. These stain colors are pic

 Core: Biology

Development and Reproduction of the Laboratory Mouse

JoVE 5159

Successful breeding of the laboratory mouse (Mus musculus) is critical to the establishment and maintenance of a productive animal colony. Additionally, mouse embryos are frequently studied to answer questions about developmental processes. A wide variety of genetic tools now exist for regulating gene expression during mouse embryonic and postnatal development, which can help…

 Biology II

An Introduction to Organogenesis

JoVE 5334

Organogenesis is the process by which organs arise from one of three germ layers during the later stages of embryonic development. Researchers studying organogenesis want to better understand the genetic programs, cell-cell interactions, and mechanical forces involved in this process. Ultimately, scientists hope to use this knowledge to create therapies and artificial organs that will help…

 Developmental Biology

An Overview of Epigenetics

JoVE 5549

Since the early days of genetics research, scientists have noted certain heritable phenotypic differences that are not due to differences in the nucleotide sequence of DNA. Current evidence suggests that these “epigenetic” phenomena might be controlled by a number of mechanisms, including the modification of DNA cytosine bases with methyl groups, the addition…

 Genetics

Transcription Factors

JoVE 10983

Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of certain tissues or body parts without affecting the entire organism. An additional layer of complexity is added by transcription factors in eukaryotes exerting combinatorial control. That means input provided by several transcription factors synchronously regulate the expression of a single gene. The combination of several transcriptional activators and repressors enables a gene to be differentially regulated and adapt to a variety of environmental changes without the need for additional genes.

 Core: Biology

An Introduction to Developmental Neurobiology

JoVE 5207

Developmental neuroscience is a field that explores how the nervous system is formed, from early embryonic stages through adulthood. Although it is known that neural progenitor cells follow predictable stages of proliferation, differentiation, migration, and maturation, the mechanisms controlling the progression through each stage are incompletely understood. Studying…

 Neuroscience

Operons

JoVE 10984

Prokaryotes can control gene expression through operons—DNA sequences consisting of regulatory elements and clustered, functionally related protein-coding genes. Operons use a single promoter sequence to initiate transcription of a gene cluster (i.e., a group of structural genes) into a single mRNA molecule. The terminator sequence ends transcription. An operator sequence, located between the promoter and structural genes, prohibits the operon’s transcriptional activity if bound by a repressor protein. Altogether, the promoter, operator, structural genes, and terminator form the core of an operon. Operons are usually either inducible or repressible. Inducible operons, such as the bacterial lac operon, are normally “off” but will turn “on” in the presence of a small molecule called an inducer (e.g., allolactose). When glucose is absent, but lactose is present, allolactose binds and inactivates the lac operon repressor—allowing the operon to generate enzymes responsible for lactose metabolism. Repressible operons, such as the bacterial trp operon, are usually “on” but will turn “off” in the presence of a small molecule called a corepressor (e.g., tryptophan). When tryptophan—an essential amino acid—is abundant, tryptophan binds and activates the

 Core: Biology

In ovo Electroporation of Chicken Embryos

JoVE 5156

Electroporation is a technique used in biomedical research that allows for the manipulation of gene expression via the delivery of foreign genetic material into cells. More specifically, in ovo electroporation is performed on early developing chicks (Gallus gallus domesticus) contained within their eggshells. In this procedure, DNA or knockdown constructs are first injected…

 Biology II

MicroRNAs

JoVE 10801

MicroRNA (miRNA) are short, regulatory RNA transcribed from introns—non-coding regions of a gene—or intergenic regions—stretches of DNA present between genes. Several processing steps are required to form biologically active, mature miRNA. The initial transcript, called primary miRNA (pri-mRNA), base-pairs with itself forming a stem-loop structure. Within the nucleus, an endonuclease enzyme, called Drosha, shortens the stem-loop structure into hairpin-shaped pre-miRNA. After the pre-miRNA ends have been methylated to prevent degradation, it is exported from the nucleus into the cytoplasm. In the cytoplasm, another endonuclease enzyme, called Dicer, cuts the pre-miRNA into a 21-24 nucleotide-long miRNA duplex. Then, Dicer cleaves one strand of the duplex, releasing a single strand of mature miRNA. The mature miRNA is loaded into a protein complex called RNA-induced silencing complex (RISC), which the miRNA then guides to the complementary region of its target mRNA. The extent of complementary base-pairing between miRNA and 3’ untranslated region of target mRNA determines the gene silencing mechanism. Extensive or near-perfect complementarity causes degradation of mRNA, whereas limited base-pairing inhibits translation. While silencing via mRNA degradation is irreversible, translation inhibition is reversible since stable mRNA can

 Core: Biology

Zebrafish Reproduction and Development

JoVE 5151

The zebrafish (Danio rerio) has become a popular model for studying genetics and developmental biology. The transparency of these animals at early developmental stages permits the direct visualization of tissue morphogenesis at the cellular level. Furthermore, zebrafish are amenable to genetic manipulation, allowing researchers to determine the effect of gene expression on the…

 Biology II

Development of the Chick

JoVE 5155

The chicken embryo (Gallus gallus domesticus) provides an economical and accessible model for developmental biology research. Chicks develop rapidly and are amenable to genetic and physiological manipulations, allowing researchers to investigate developmental pathways down to the cell and molecular levels.


This video review of chick development begins by describing the…

 Biology II

SNP Genotyping

JoVE 5544

Single nucleotide polymorphisms, or SNPs, are the most common form of genetic variation in humans. These differences at individual bases in the DNA often do not directly affect gene expression, but in many cases can still be useful for locating disease-associated genes or for diagnosing patients. Numerous methodologies have been established to identify, or…

 Genetics

An Introduction to the Zebrafish: Danio rerio

JoVE 5128

Zebrafish (Danio rerio) are small freshwater fish that are used as model organisms for biomedical research. The many strengths of these fish include their high degree of genetic conservation with humans and their simple, inexpensive maintenance. Additionally, gene expression can be easily manipulated in zebrafish embryos, and their transparency allows for observation of developmental…

 Biology II

Induced Pluripotency

JoVE 5333

Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed to form undifferentiated stem cells. Like embryonic stem cells, iPSCs can be grown in culture conditions that promote differentiation into different cell types. Thus, iPSCs may provide a potentially unlimited source of any human cell type, which is a major breakthrough in the field of regenerative…

 Developmental Biology

Rodent Stereotaxic Surgery

JoVE 5205

Stereotaxic (or stereotactic) surgery is a method used to manipulate the brain of living animals. This technique allows researchers to accurately target deep structures within the brain through the use of a stereotaxic atlas, which provides the 3D coordinates of each area with respect to anatomical landmarks on the skull. After the skull is exposed, anesthetized animals…

 Neuroscience

Intracellular Hormone Receptors

JoVE 10876

Lipid-soluble hormones diffuse across the plasma and nuclear membrane of target cells to bind to their specific intracellular receptors. These receptors act as transcription factors that regulate gene expression and protein synthesis in the target cell

Based on their mode of action, intracellular hormone receptors are classified as Type I or Type II receptors. Type I receptors, including steroid hormone receptors such as the androgen receptor, are present in the cytoplasm. Hormone binding transports the hormone-receptor complex to the nucleus, where it binds to regulatory DNA sequences called hormone response elements and activates gene transcription. Type II receptors, such as the thyroid hormone receptor, are bound to their DNA response elements within the nucleus even in the absence of hormone. In this state, the receptor acts as an active repressor of transcription. However, upon hormone binding, the receptor-hormone complex activates transcription of thyroid hormone-inducible genes.

 Core: Biology

Isolating Nucleic Acids from Yeast

JoVE 5096

One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.


This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps…

 Biology I

PCR

JoVE 10819

The polymerase chain reaction, or PCR, is a widely used technique for copying segments of DNA. Due to exponential amplification, PCR can produce millions or billions of DNA copies within just a few hours. In a PCR reaction, a heat-resistant DNA polymerase enzyme amplifies the original DNA through a series of temperature changes inside an automated machine called a thermocycler.

Kary Mullis developed PCR in 1983, for which he was awarded the 1993 Nobel Prize in Chemistry. Being a relatively fast, inexpensive, and precise way of copying a DNA sequence, PCR became an invaluable tool for numerous applications, including molecular cloning, gene mutagenesis, pathogen detection, gene expression analysis, DNA quantitation and sequencing, and genetic disease diagnosis. PCR mimics the natural DNA replication process that occurs in cells. The reaction mixture includes a template DNA sequence to be copied, a pair of short DNA molecules called primers, free DNA building blocks called deoxynucleotide triphosphates (dNTPs), and a specialized DNA polymerase enzyme. PCR involves a series of steps at high temperatures, requiring a DNA polymerase enzyme that is functional at such temperatures. The most commonly used DNA polymerase is Taq polymerase, named after Thermus aquaticus, the bacterium from which the polymerase was initially isolated. DNA

 Core: Biology

Murine In Utero Electroporation

JoVE 5208

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although…

 Neuroscience

Transplantation Studies

JoVE 5336

Many developmental biologists are interested in the molecular signals and cellular interactions that induce a group of cells to develop into a particular tissue. To investigate this, scientists can use a classic technique known as transplantation, which involves tissue from a donor embryo being excised and grafted into a host embryo. By observing how transplanted tissues develop in host…

 Developmental Biology

Zebrafish Microinjection Techniques

JoVE 5130

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is…

 Biology II

Complementary DNA

JoVE 10818

Only genes that are transcribed into messenger RNA (mRNA) are active, or expressed. Scientists can, therefore, extract the mRNA from cells to study gene expression in different cells and tissues. The scientist converts mRNA into complementary DNA (cDNA) via reverse transcription. Because mRNA does not contain introns (non-coding regions) and other regulatory sequences, cDNA—unlike genomic DNA—also allows researchers to directly determine the amino acid sequence of the peptide encoded by the gene. cDNA can be generated by several methods, but a common way is to first extract total RNA from cells, and then isolate the mRNA from the more predominant types—transfer RNA (tRNA) and ribosomal (rRNA). Mature eukaryotic mRNA has a poly(A) tail—a string of adenine nucleotides—added to its 3’ end, while other types of RNA do not. Therefore, a string of thymine nucleotides (oligo-dTs) can be attached to a substrate such as a column or magnetic beads, to specifically base-pair with the poly(A) tails of mRNA. While mRNA with a poly(A) tail is captured, the other types of RNA are washed away. Next, reverse transcriptase—a DNA polymerase enzyme from retroviruses—is used to generate cDNA from the mRNA. Since, like most DNA polymerases, reverse transcriptase can add nucleotides only to the 3’ end of a chain, a pol

 Core: Biology

Types of Hormones

JoVE 10988

Hormones can be classified into three main types based on their chemical structures: steroids, peptides, and amines. Their actions are mediated by the specific receptors they bind to on target cells.

Steroid hormones are derived from cholesterol and are lipophilic in nature. This allows them to readily traverse the lipid-rich cell membrane to bind to their intracellular receptors in the cytoplasm or nucleus. Once bound, the cytoplasmic hormone-receptor complex translocates to the nucleus. Here, it binds to regulatory sequences on the DNA to alter gene expression. Peptide hormones are made up of chains of amino acids and are hydrophilic. Hence, they are unable to diffuse across the cell membrane. Instead, they bind to extracellular receptors present on the surface of target cells. Such binding triggers a series of signaling reactions within the cell to ultimately carry out the specific functions of the hormone. Amine hormones are derived from a single amino acid, either tyrosine or tryptophan. This class of hormones is unique because they share their mechanism of action with both steroid as well as peptide hormones. For example, although epinephrine and thyroxine are both derived from the amino acid tyrosine, they mediate their effects through diverse mechanisms. Epinephrine binds to G-protein coupled receptors present on the surface of the plasma

 Core: Biology

Intracellular Signaling Cascades

JoVE 10721

Intracellular signaling cascades amplify a signal originating extracellularly and directs it to its intended intracellular target resulting in transcription, translation, protein modifications, enzyme activation, cellular metabolism, mitosis, and/or apoptosis.

The most basic of signaling cascades involves the activation of second messengers and the release of kinases. Kinases activate or deactivate proteins and enzymes by adding a phosphate group to them. Phosphatases remove phosphate groups resulting in the deactivation or reactivation of proteins. The cyclic AMP (cAMP) pathway is named for its second messenger, cAMP. This pathway is most often initiated when a ligand binds to a G-coupled protein receptor. The G-protein decouples from the receptor and triggers adenylate cyclase to synthesize cAMP from ATP. For each ligand-receptor interaction, multiple cAMP molecules are generated—amplifying the signal. cAMP activates protein kinase A (PKA). PKA is a tetramer molecule with two regulatory subunits and two active subunits. When four cAMP molecules interact with a PKA molecule, it releases the two active subunits. These PKA subunits phosphorylate target proteins and enzymes. In the case of gene expression, PKA activates CREB, a transcription factor in the nucleus. The steps that precede the intracellular signaling cascade that is the lig

 Core: Biology
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