Cell Biology and Histology - Medical School Crash Course

Cell Biology &

Histology Medical School Crash Course™

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Table of Contents Preface ................................................................................................................................... 7 Chapter 1: Introduction to Medical Cell Biology ...................................................................... 9 Molecular Biology ....................................................................................................................... 9 Cell Biology Origins ................................................................................................................... 10 Cell Biology Basics ..................................................................................................................... 11 Biological Molecules ................................................................................................................. 13 Histogenesis and Germ Layers .................................................................................................. 13 Ectodermal Cells ....................................................................................................................... 14 Medical Conditions associated with Ectoderm Failure............................................................. 14 Mesodermal Cells...................................................................................................................... 15 Endodermal Cells ...................................................................................................................... 16 Cell Biology Techniques ............................................................................................................ 16 Blotting Techniques .................................................................................................................. 17 DNA Microarray Technique ...................................................................................................... 18 Key Takeaways .......................................................................................................................... 18 Quiz ........................................................................................................................................... 18 Chapter 2: Histology Basics ................................................................................................... 22 Histology and Histopathology ................................................................................................... 22 Normal Tissues .......................................................................................................................... 22 Histology Preparation ............................................................................................................... 23 Gram Staining Technique .......................................................................................................... 27 Miscellaneous Stains and Techniques ...................................................................................... 29 Typical Biological Stains ............................................................................................................ 30 Histochemistry .......................................................................................................................... 31 Dealing with Artifacts................................................................................................................ 31 Summary of Tissue Collection and Examination....................................................................... 32 Key Takeaways .......................................................................................................................... 33 Quiz ........................................................................................................................................... 33 Chapter 3: Cell membranes ................................................................................................... 36 Cell Membrane Basics ............................................................................................................... 36

Cell Membrane Transport ......................................................................................................... 38 The Lipid Bilayer of the Membrane .......................................................................................... 39 Carbohydrates in the Cell Membrane....................................................................................... 41 Proteins in the Cell Membrane ................................................................................................. 41 Cell Membrane Receptors ........................................................................................................ 42 Mechanism of Action ................................................................................................................ 43 Understanding Receptor-based Diseases ................................................................................. 44 Key Takeaways .......................................................................................................................... 45 Quiz ........................................................................................................................................... 45 Chapter 4: Cytoskeleton........................................................................................................ 49 Cytoskeleton Basics................................................................................................................... 49 Actin Filaments ......................................................................................................................... 50 Microtubules ............................................................................................................................. 52 Intermediate Filaments............................................................................................................. 55 Septins ....................................................................................................................................... 56 Cytoskeleton Diseases .............................................................................................................. 57 Key Takeaways .......................................................................................................................... 57 Quiz ........................................................................................................................................... 58 Chapter 5: Organelle Structure and Function ........................................................................ 61 Nucleus...................................................................................................................................... 61 Nuclear Envelope ...................................................................................................................... 62 Mitochondria ............................................................................................................................ 62 Nucleolus................................................................................................................................... 63 Cytosol....................................................................................................................................... 63 Ribosome .................................................................................................................................. 64 Lysosomes ................................................................................................................................. 66 Centrosomes ............................................................................................................................. 67 Peroxisomes .............................................................................................................................. 67 Endoplasmic Reticulum............................................................................................................. 67 Golgi Apparatus......................................................................................................................... 69 Key Takeaways .......................................................................................................................... 70

Quiz ........................................................................................................................................... 70 Chapter 6: Gene Expression and Translation ......................................................................... 73 Gene Expression........................................................................................................................ 73 Transcription ............................................................................................................................. 74 Translation ................................................................................................................................ 75 Protein Folding .......................................................................................................................... 76 Key Takeaways .......................................................................................................................... 77 Quiz ........................................................................................................................................... 77 Chapter 7: Cell Adhesion and the Extracellular matrix ........................................................... 81 Cell Adhesion ............................................................................................................................ 81 Pemphigus................................................................................................................................. 82 Extracellular Matrix (ECM) ........................................................................................................ 83 Types of Proteoglycans in the ECM .......................................................................................... 85 Proteins in the ECM .................................................................................................................. 86 Miscellaneous ECM Components ............................................................................................. 87 The ECM and Tissue Regeneration ........................................................................................... 88 Key Takeaways .......................................................................................................................... 88 Quiz ........................................................................................................................................... 89 Chapter 8: Intercellular signaling........................................................................................... 93 Intercellular Recognition........................................................................................................... 93 Cell Junctions ............................................................................................................................ 93 Non-Junctional Cell Signaling .................................................................................................... 95 Signal Molecules and Signal Receptors..................................................................................... 95 Stages of Intercellular Communication .................................................................................... 96 Key Takeaways .......................................................................................................................... 97 Quiz ........................................................................................................................................... 97 Chapter 9: Cell division, the Cell Cycle, and Cancer .............................................................. 101 Cell Division ............................................................................................................................. 101 Phases of Cell Division............................................................................................................. 101 Cellular Differentiation ........................................................................................................... 102 Cell Categories and Cell Types ................................................................................................ 103

Cancer and Cell Division .......................................................................................................... 104 Key Takeaways ........................................................................................................................ 106 Quiz ......................................................................................................................................... 106 Chapter 10: Programmed Cell Death and Autophagy .......................................................... 109 Apoptosis ................................................................................................................................ 109 Apoptotic Intrinsic Pathway.................................................................................................... 110 Extrinsic Pathway of Apoptosis ............................................................................................... 111 Dead Cell Removal .................................................................................................................. 113 Apoptosis and Disease ............................................................................................................ 113 Viral Infections and Apoptosis ................................................................................................ 114 Autophagy ............................................................................................................................... 114 Key Takeaways ........................................................................................................................ 115 Quiz ......................................................................................................................................... 115 Summary ............................................................................................................................ 119 Course Questions ................................................................................................................ 121 Course Answers .................................................................................................................. 141

Preface This course is intended to be a thorough discussion of most of the important areas of study connected with cell biology. Cell biology happens on a molecular level and on a microscopic level, with research being an active part of both aspects of cell biology. In the first chapter, the different types of cells will be discussed along with the basic techniques that cell biologists use to study cellular features in medicine and in the study of diseases directly connected to cellular dysfunction.

In the second chapter, the topic of histology will be discussed. Much of the practice of histology involves histopathology, which is the microscopic study of diseased tissues and organs. The ways in which pathologists take specimens and study them under the microscope will be covered as part of this chapter as well the importance of histology in medical and surgical practice.

In the third chapter, the structure and function of the cell membrane will be the topic of discussion. You will discover that the cell membrane is more than just a lipid bilayer. It has a vital function to the cell and contains proteins, carbohydrates, and other molecules that make the cell membrane a dynamic part of cellular metabolism and cellular function.

In chapter four, the cytoskeleton will be the main topic of discussion. The cytoskeleton is made from several types of filaments, some of which are designed for movement of the cell, while others are static and provide just the structure of the cell. The characteristics of these filaments from a molecular standpoint are intended to be a large feature of this chapter.

Chapter five will be a basic discussion of the various organelles in the cell. The organelles consist of things like the nucleus, the nucleolus, the endoplasmic reticulum, the Golgi apparatus, the lysosomes, the ribosomes, and the peroxisomes. They work independently and together to create a metabolically-active cell. How they work together will be covered in this chapter.

Chapter six is intended to be a thorough discussion of gene expression and the translation of genetic material into active proteins. This is a complex process involving several organelles and various molecules. Each cell in the human body contains the entire human genome but only a few genes are expressed at any given point in time. These genes get transcribed as the beginning step that ultimately leads to the translation of proteins.

Chapter seven will cover the basics of cell adhesion and the importance of the extracellular matrix. Cells must hold together to form a complete tissue and a complete organ. The connections between cells vary according to the tissue and the extracellular matrix also varies, depending on the type of tissue involved.

The subject of intercellular signaling is the topic of chapter eight. Cells must communicate with one another to function as a complete organ. This is all done on a molecular level, and involves local cellular connections in some cases and things like hormones in other cases, that deliver cellular messages over great distances. In this chapter, both local and distant intercellular communication will be discussed.

Things like cell division, the cell cycle, and how these relate to cancerous cells are intended to be the major topic of chapter nine. Cell division happens to nearly all cells of the body in a controlled way; however, in cancer cells, cell division doesn’t have any stopgaps and there are no mechanisms for the programmed cell death of these types of cells, making them very dangerous for the organism.

Lastly, the tenth and concluding chapter of the course will involve a discussion of programmed cell death, which is also called apoptosis. It is a normal part of an organism’s need to get rid of cells that have become damaged or have aged to a certain point in the organism’s life.

Autophagy is related to apoptosis but involves the digestion of certain parts of the cell that are no longer needed. These parts of the cell are digested in an organized way with recycling of the components that get reused by the cell in an evolutionary, efficient way.

Chapter 1: Introduction to Medical Cell Biology Cell biology is the study of cells, how they function, how they divide, and how they communicate with each other. In this first chapter, the basics of molecular biology, cell biology, and the techniques used to study cells are discussed. Part of this course will also discuss the medical implications of cell biology, including diseases that originate from abnormalities of the cell.

Molecular Biology Molecular biology is the study of the molecules involved in cell function. Molecules that participate in cell function are called biomolecules, which are biologically active and work together to make the cell function. Examples of biomolecules are DNA, RNA, proteins, lipids, and carbohydrates. Enzymes are biomolecules that have kinetic activity, which is used to drive biochemical reactions. Many diseases in the human body come from enzyme deficiencies that result in cellular structural and functional problems, leading to disease states that usually involve the entire body.

Molecular biology does not exist in a vacuum but its study is closely related to the study of the biochemistry of cells and the genetic functions of the cells. Without biochemistry and biochemical reactions, cellular functions would cease and cells would die. Without genetics, DNA and RNA would not exist, and the transcription of proteins and enzymes necessary for life would not happen. Researchers used ideas and techniques from both biochemistry and genetics to understand the molecular cell biology reactions that need to happen to have cellular life.

Biochemistry as a discipline is the study of biologically-active chemical substances and the vital reactions that occur in living organisms. The role of biomolecules, such as enzymes, proteins, phospholipids, and genetic molecules are a big part of what is studied in biochemistry.

Genetics also relates strongly to cell molecular biology. The DNA of each cell is the same; however, some genes are turned on in certain cells and turned off in other cells, giving cells their ability to be different from one another. Remember that all cells ultimately come from a single cell with all the DNA necessary to make a living being but that, for the living being to develop, the DNA must translate different proteins in different cells so that the cells can be unique from one another as they multiply and divide in the embryo, fetus, infant, and more grown-up human beings.

Genetics also is the study of the genetic differences in organisms. Genes code for proteins in each cell; the proteins might be structural proteins or enzymatic proteins, depending on the cell needs. As the enzymes make the different biochemical reactions, a certain phenotype appears.

The phenotype of a cell is not much different from the phenotype of the organism. The cell’s phenotype is the actual appearance of the cell, which ultimately determines the appearance of

the tissue the cells make together, and finally, the organs and phenotype of the human, plant, or animal that is visible to the outside world.

Molecular biology, then, refers to the study of cellular replication, cell function, transcription of genes, and translation of genetic material into proteins, enzymes, and other molecules. The central focus of molecular biology is how DNA creates transcription RNA that goes on in the cell to make the important proteins necessary for cell function. One subfield of molecular biology is molecular genetics, which studies the DNA molecule, how DNA and RNA work together, and how genetic diseases can be defined as mutations in the genetic code of the various cells of the body.

Cell Biology Origins As mentioned, all of cell biology starts with the fusion of the sperm and egg in the fertilization process. When this happens, the result is a zygote, which is an alternate term for the fertilized egg. As this cell divides, it forms a cluster of cells that are all similar in nature, called a blastula.

The divisions of this original cell happen quite quickly with no cellular growth so that the daughter cells are smaller in size than the original cell and the embryo does not increase in size.

This type of cell division is referred to as cleavage divisions.

Figure 1 shows the different germ layers:

Figure 1

Three separate cell layers begin to form from the blastula that allow for the differentiation of cells that ultimately form the being. These germ layers or sheets are referred to as the ectoderm, the mesoderm, and the endoderm. Each type of germ layer forms multiple unique cells that together form the embryo, fetus, and infant. This is the first type of specification that forms in the growing blastula. Besides the three germ layers, cells that begin to create the mammalian placenta begin to form, which is the structure than nourishes the embryo. The placenta does not come from any of the germ layers.

As the embryo develops, some cells turn on specific genes, while other cells turn off genes, which results in the specification of cells. The upregulation of specific genes makes proteins in the cytoplasm that do different things inside the cell, creating the molecules and organelles necessary for cell function. The result of the upregulation of certain genes and the downregulation of certain genes is the formation of different cell types that go from the simplest cell types—the ectodermal cells, endodermal cells, and mesodermal cells—to the more complex cells, like nerve cells, muscle cells, and epidermal cells.

As the process of differentiation of the blastula occurs, the ectodermal cells arrive on the outside of the blastula, the mesodermal cells arrive at the middle of the blastula, and the endodermal cells arrive at the inside of the blastula/embryo. These cells communicate with one another, beginning the process of intracellular communication, which will be discussed in chapter 8. The shape of the embryo begins to form with a caudal aspect, mesial aspect, and cephalad aspect, giving the tail, trunk, and head of the embryo, respectively.

Rather quickly, the embryo begins to grow because different DNA molecules are turned on in certain cells that change the growth rate of each specific type of cell. Some cells grow faster than others, which creates the unique shape of the embryo. The head is larger than the tail because these cells grow faster and bigger than the caudal cells so the embryo takes on the shape of a larger head, a medium-sized middle, and a smaller tail. No one knows how these cells communicate with one another to create this unique shape.

Cell Biology Basics The cellular unit consists of numerous different molecules that make organelles—each of which is enclosed by a membrane of some sort.

The organelles are separate from one another but are connected by an intracellular matrix that allows them to stay basically in the same place unless they are needed to travel to one or another part of the cell.

Refer to figure 2 to see what the typical cell looks like:

Figure 2 Cells need to both divide and grow. They can’t do this without some sort of basic cellular nutrition and cellular energy. The cell uses organelles that ingest molecules from outside the cell, using them inside the cell for cellular growth and development. The molecules ingested by the cell are acted upon by enzymes that catalyze biochemical reactions. The biochemical reactions can create cell structures or can be part of the energy pathways necessary for the cell to function. All biochemical reactions in the cells use catalysts to make the reaction happen.

The catalyst is necessary for the reaction but doesn’t change during the reaction so it is available for further reactions.

Cellular processes and cellular reactions are not singular events but occur in chains of reactions that go through multiple steps to take a single molecule and, over many steps, transform it into the desired molecule necessary for the cell’s function. In general, small molecules are ingested and, through multiple enzymatic steps, are transformed into bigger molecules that make cell structures. When there are enough big molecules inside the cell to support cell division, the process of cell division occurs, creating two daughter cells that have enough substrate molecules to support themselves. This process happens millions of times to create cells of all types and sizes.

Biological Molecules The basic component of every living cell is the biological molecule. Most biological molecules are carbon-based, meaning that the carbon atom forms the basic structure of the cell. Carbon atoms are particularly suited as molecular basics because they can form stable biochemical bonds with four different atoms. Chains and rings of carbon-based molecules contain oxygen molecules, hydrogen molecules, and nitrogen molecules—which together form arrays of molecules that can contain as little as ten atoms or as many as millions of atoms all linked together in a specific pattern.

There are four types of organic biological molecules. They are sugars (carbohydrate basics), amino acids (protein basics), nucleotides (DNA and RNA basics), and fatty acids (lipid basics).

Each type of organic molecule has a specific shape and function based on what types of atoms are attached to the carbon-based chain. Large macromolecules are ultimately made from these simple and basic substrates. In cells, the primary carbohydrate is glycogen, while the primary lipid is the organelle/cell membrane. The primary form of nucleotide-based molecules is the chromosome. Proteins are about equally divided into structural proteins and enzymatic proteins.

The majority of the mass of a typical mammalian cell is water, which makes up seventy percent of a cell’s mass. The rest are made from macromolecules, of which proteins make up the largest mass. The average size of a given protein is four hundred amino acid molecules long and, based on which amino acids make up the protein, the protein has a specific shape. An infinite number of protein molecules can be made from the basic twenty amino acids used to make these molecules. It is necessary that the protein fold in a specific way to be functional. There are some genetic diseases affecting the coding for various proteins so that they don’t fold properly and don’t function in the way they are supposed to fold.

Most of the catalysts inside mammalian cells are enzymes and most enzymes are entirely made from proteins. The major key to the ability of the enzyme to function is its unique shape, which allows it to bind to a molecule and transform it into a different molecule. Ribozymes are also catalysts that have a unique shape. They consist of lines of nucleotides folded in a way that allows them to make chemical reactions. Ribozyme-catalyzed reactions are less common than enzyme-catalyzed reactions because there are many more combinations that can be created from amino acids than from nucleotides, of which there are only a few types.

Histogenesis and Germ Layers Ultimately, the undifferentiated cells must become differentiated to form the numerous kinds of cells in the mammalian body. The first differentiation that occurs has already been mentioned, which is the differentiation into the endoderm, mesoderm, and ectoderm. The science that describes how these three germ layers turn into multiple types of cells is called “histology”, which is the main topic discussed in chapter two of this course.

A germ layer is nothing more than a specific collection of cells formed during embryogenesis.

Other types of organisms will form only a couple of germ layers but all mammals and almost all vertebrates, including humans, begin with the basic three layers that differentiate even further into tissues and organs.

Ectodermal Cells Ectodermal cells form one of the main germ layers in the earliest embryonic stages. It is the outermost layer of the blastocyst, forming multiple types of cells. For example, it is the ectodermal cells that form the skin of the human, the nervous system (including the spine, the brain, and the peripheral nervous system. The mouth, sweat glands, nostrils, nails, hair, and tooth enamel all originate from ectodermal cells. There are three parts to the ectoderm in humans: the external ectoderm (surface ectoderm or the skin), the neural crest, and the neural tube (spinal cord).

The first appearance of the embryo is a spherical, hollow blastula, with the ectoderm being on the outside layer of this sphere. Ectodermal cells have selective affinity for the mesoderm but not for the endoderm, which means the ectoderm is connected to the mesoderm during embryonic development.

It is the process known as gastrulation, which begins with the formation of bottle cells that punch a hole into the blastula surface and extend inward to from the roof of the blastocoel. The mesoderm cells migrate into the blastocyst, while the ectodermal cells stay on the outside, forming a thin layer that becomes the skin. After the process of gastrulation finishes, the ectoderm is on the outside, the mesoderm is in the middle, and the endoderm is on the inside.

The ectoderm then begins to differentiate in a process known as neurulation, which forms the epidermis, the neural crest, and the neural tube. Each of these, in turn, become specialized cells. The neural tube cells become the central nervous system cells, the neural crest cells become the enteric and peripheral nervous system, the melanocytes, the facial cartilage, and the teeth dentin, while the epidermal cells become the epithelium of the mouth, the eyes, the sebaceous glands, the olfactory epithelium, the skin, hair, and nails.

There are two parts to neurulation: primary and secondary neurulation, which position the neural crest cells in the middle of the epidermal cells and the deep neural tube. There are notochord cells in the mesoderm that are connected to superficial ectoderm cells that tell those cells to form a columnar neural plate. They elongate and change shape, becoming medial hinge cells. These cells fold inward by massive amounts of cell division to form dorsolateral hinge cells. The result is a hollow, neural tube that is surrounded by epidermis.

Medical Conditions associated with Ectoderm Failure There are almost two hundred types of ectodermal dysplasia diseases—all of which are very rare. They involve abnormal development of any one or more of the structures of the

ectodermal cells because of a genetic defect that happens early in development. The most common type of ectodermal dysplasia is called hypohidrotic ectodermal dysplasia or HED. Patients with HED are unable to sweat because the ectoderm never formed sweat cells. Some of these patients also have disfigurement of the face and missing teeth, wrinkling of the skin near the eyes, thin hair, and a misshapen nose. This is an X-linked recessive disease affected males almost exclusively. Females, however, can be carriers of the disease.

Mesodermal Cells The mesoderm is the middle embryonic layer, which is packed between the endoderm layer and the ectoderm layer. It forms non-epithelial blood cells and muscle cells. It forms the mesentery of the human/mammal and the part of the gonads that is not made directly from gametes. Like other aspects of differentiation, the formation of the various organs relies on intercellular signaling that polarizes cells into different areas of the embryo. Besides making these structures, the mesoderm is the inducing factor for the development of the neural plate (which is ectodermal-derived tissue).

The separation of the embryo into the three different layers and the process of gastrulation occurs in the third embryonic week. During this process, three types of mesoderm are produced: paraxial mesoderm, intermediate mesoderm, and lateral plate mesoderm. Each has its own function. Paraxial mesoderm forms the bones, cartilage, and subcutaneous skin tissue.

Cells called somites are responsible making these tissues and are turned on by nearby embryonic structures, such as the epidermis, neural tube, and neural cord.

The intermediate mesoderm gives rise to the urogenital organs of the body, including the gonads (that aren’t gametes), kidneys, adrenal glands, and the ducts that connect these structures. The lateral plate mesodermal cells make the heart, the blood vessels, and the blood cells. Parts of the limbs deep to the skin are created from mesoderm. All muscle types come from mesoderm, including skeletal muscle, cardiac muscle, and smooth muscle. Connective tissue, bone, cartilage, subcutaneous fat, blood cell endothelium, white blood cells, kidneys, adrenal cortex, and red blood cells all come from mesoderm.

The notochord production is under the control of the mesoderm, forming the neural tube and the linear axis of the embryo, extending from the head of the embryo to the tail of the embryo.

The part of the mesoderm that forms the paraxial mesoderm forms the covering over the notochord. The process of making the notochord happens between the fifteenth and seventeenth day of embryonic development.

The paraxial mesoderm is intimately connected to spinal cord development. It is these cells that grow up and down the embryo, covering the neural structures and ultimately forming the backbone or axial skeleton. There are different somites in the mesoderm that differentiate into their own compartment that contains specific muscles, cartilage, and tendons—each of which is serviced by a different dermatome that comes from the ectoderm.

As mentioned, multiple different somites from the mesodermal germ layer separate into the various bones of the spinal column. There is notochord protein that builds up in the mesoderm, allowing the somites to be established. Protein SHH is activated by the cells of the notochord

and the neural tube that helps make the individual mesodermally-derived sclerotome. The sclerotome cells make protein PAX1 that turns on the formation of bone and cartilage in the spine. Protein WNT1 is activated by the neural tube, turning on PAX2, which regulates the formation of the dermatome and myotome. Lastly, NT-3 is a protein made by the neural tube that tells the mesodermal somite to make dermis. Initially, the formation of these structures is completely symmetric so the left side equals the right side but, eventually, there are differences between the muscles of the left side of the body and the muscles of the right side of the body.

There are two components to the lateral plate mesoderm, which include the parietal (somatic) layer and the visceral (splanchnic) layer. These start to make the body’s cavities. The somatic layer covers the amnion and the visceral layer covers the yolk sac—both of which line the intraembryonic cavity, forming the lateral folds of the body wall. The visceral layer eventually lines the gut, while the parietal layer lines the pericardial, pleural, and peritoneal cavities.

Endodermal Cells The endoderm is the innermost layer of the three main germ layers in the embryo. During the gastrulation process, these cells migrate inward, forming the inner lining of the blastocyst ball.

The cells are flat and columnar, making them perfectly shaped to become the epithelial lining of many different body systems. The two main tubes made with endodermal cells are the digestive tube and the respiratory tube, although there are other systems that rely on the ectoderm.

In the gastrointestinal tube, these cells line the entire alimentary canal except for the rectum (which is derived from ectoderm), and parts of the mouth and pharynx (which are also ectoderm-derived). The cells lining the liver and pancreas come from endodermal cells. In the respiratory tube, the entire lining of the lungs, alveoli, bronchi, and trachea come from endodermal cells. The follicular lining of the thyroid gland and the epithelial lining of the thymus come from endoderm. The urinary bladder, part of the urethra, the auditory tube, and the tympanic cavity are all lined by endoderm-derived cells.

Cell Biology Techniques There are multiple techniques used by scientists that are based on an understanding of and a use of cellular biology. The first is molecular cloning. This technique depends on finding a DNA segment that codes for a protein and isolating the DNA. The DNA is cloned so that millions of copies of the DNA are made and are placed inside expression vectors called plasmids. Plasmids are usually resistant to antibiotics, have multiple DNA cloning sites, and a specific place allocated for replication.

The plasmid is inserted into an animal cell or a bacterial cell and begins making copies of the intended protein. The insertion techniques vary but can involve a viral vector that gets into the cell or by means of uptake of naked DNA into the cell. The introduction of DNA into an animal cell for the purposes of cloning is known as transfection. After transfection has occurred, the DNA can remain as it is or can be incorporated into the genome of the cell. If the DNA is independent, it is called “transient transfection” and if the DNA is incorporated into the genome, it is called “stable transfection”.

What happens then is that the protein is expressed by the animal or bacterial cell. It is expressed in high levels because multiple copies of the gene are inserted so that a great deal of the desired protein can be extracted from the bacterial cell or animal cell. It can be tested for enzymatic activity if it is an enzymatic protein and can be crystallized to make a drug that could be useful to the pharmaceutical companies. Theoretically, patients with an enzyme deficiency could be inoculated with a plasmid containing the missing enzyme and could be cured because they now have the DNA necessary to make the missing enzyme.

Another commonly-used technique in molecular cell biology is the polymerase chain reaction or PCR. This is a way of copying sequences of DNA. The technique is so successful that more than a billion DNA molecules can be isolated from a single strand of DNA in under two hours. The technique can be used to create mutations on existing DNA or to add sections of DNA coding for specific enzymes on the ends of normal chromosomes. Mutating DNA by this method is called “site-directed mutagenesis”. There are different PCR techniques that can identify amplify both DNA and RNA, making protein synthesis possible.

Molecular cell biologists also make use of gel electrophoresis. The idea is the different lengths of DNA, proteins, and RNA migrate differently along an electrical field. Gel is used and strands of DNA are placed on one end of the gel. An electrical field is applied to the gel and the strands migrate a certain distance, depending on their size and electrical charge. These can be stained and identified by laboratory scientists as ways to find certain strands of DNA, RNA, or proteins.

Blotting Techniques There are four different blotting techniques used by molecular and cell biologists. The first is the Southern blot. It is a technique that probes for a certain DNA strand on a sample of DNA. The DNA is sometimes digested by restriction enzymes (which is also called restriction endonuclease digestion) and is then separated using gel electrophoresis. The DNA is taken up by a membrane by means of capillary action before being exposed to a labeled probe that matches the DNA segment of interest. If the DNA segment of interest is present, it will stain appropriately. This technique is less effective than PCR so it isn’t used as often as it used to be.

The Northern blot technique is used to understand expression patterns of different types of RNA molecules compared to a standard set of RNA molecules. This combines gel electrophoresis of RNA and a blotting technique. RNA is separated by the gel procedure and placed on a membrane, where it is labeled to identify a sequence of interest. It is a test that measures gene expression because genes that are being expressed will make RNA, while genes that aren’t being expressed won’t make RNA. Western blotting techniques involve the separation of proteins by gel electrophoresis by size and electrical charge. The proteins are then placed between two glass plates and transferred to membranes that can be probed with antibodies that are stained or otherwise tagged. This procedure sometimes uses chemiluminescence and enzymes to allow for the qualitative and quantitative evaluation of certain proteins in tissue or cell sections.

Eastern blotting also measures proteins but it measures the post-translational modification of proteins. Substrates are used to probe for proteins that have been blotted onto membranes

after being modified. In other ways, this technique strongly resembles the other blotting techniques already described.

DNA Microarray Technique A DNA microarray is another DNA detection method. DNA is separated into a collection of spots that are affixed to a microscope slide. Each spot represents at least tone single short strand of DNA. Large amounts of DNA spots can be placed on a single slide and each DNA spot is representative of a specific DNA sequence that can be identified. It can be varied to determine the expression of genes by doing the same technique on RNA in a tissue. The RNA is converted to a labeled form of DNA that is fragmented and placed on a slide. Those DNA segments that were expressed and made RNA will show up as being labeled spots on the slide. Multiple arrays can be done on both healthy and cancerous tissue to identify which genes are being expressed on the cancerous tissues when compared to the healthy tissues. It can tell how genes are expressed over time or how they are expressed by different factors. Microscope slides and silicon chips are used to make microarrays or macroarrays, which have more than ten thousand different DNA spots on a single array. Similar arrays can be done with proteins and other nonDNA molecules to identify them.

The allele specific oligonucleotide technique is another test used by cell biology scientists. It allows for the detection of a mutation of a single base in a DNA sequence without having to use gel electrophoresis or PCR. Short radiolabeled probes are exposed to target DNA that has not been degraded. The target DNA is then washed so only the probes that have hybridized with the DNA sample will remain with the sample. Fluorescent-labeled probes can also be used.

After washing, the pieces of DNA that have hybridized with the probe are identified and single mutations can be isolated.

Key Takeaways •

All animal species begin with the fertilized egg.

•

Cell lines for all cells come from ectoderm, mesoderm, or endoderm germ layers

•

Molecular cell biologists use multiple techniques to identify DNA mutations or to grow multiple copies of a given protein in a cell line

Quiz 1. You are studying molecular biology and recognize that what type of biomolecule has kinetic activity that drives biochemical reactions? a. Phospholipid b. Lipoprotein c. Deoxyribonucleic acid d. Enzyme

Answer: d. An enzyme can be defined as a biomolecule with kinetic activity that drives biochemical reactions inside the cell.

2. In studying molecular cell biology, what term best defines the structural appearance of a cell, tissue, organ, or living being? a. Genotype b. Phenotype c. Biochemistry d. Haplotype Answer: b. The phenotype is the structural appearance of a cell, a tissue, an organ, or a living being that is ultimately determined by the genotype. The biochemical reactions together create the structure that is seen by the human eye or by a microscope.

3. When the sperm and egg unite to form a fertilized egg, the result is a single cell that is also referred to as what? a. Blastula b. Embryo c. Fetus d. Zygote Answer: d. The alternate name for the single-celled fertilized egg is the zygote. As it divides, it forms the blastula and then the embryo. Only much later in development is the growing being called a fetus, which contains numerous differentiated cells.

4. There are substrates that make up the different types of biological molecules. Which type of substrate is responsible for the formation of proteins? a. Simple sugars b. Amino acids c. Nucleotides d. Fatty acids Answer: b. Proteins are almost always entirely made from an amino acid substrate, although other components can be part of the basic amino acid structure.

5. Besides water, which type of macromolecule makes up the majority of the cell’s mass in a mammalian cell?

a. Phospholipid b. Structural protein c. DNA d. RNA Answer: b. Structural proteins (and enzymes) together make up the majority of the macromolecules in the mammalian cell.

6. Which embryonic structure is not considered to be created from the ectoderm? a. Muscle cells b. Neural crest c. Neural tube d. Epidermis Answer: a. The neural tube, the neural crest, and the epidermis all come from the ectoderm, while the muscle cells originate from another type of embryonic germ cell layer.

7. Which embryonic germ layer ultimately gives rise to a dermatome? a. Lateral plate mesoderm b. Paraxial mesoderm c. Ectoderm d. Intermediate mesoderm Answer: c. The ectoderm forms the dermatome that gives rise to the innervation of the various myotomes that come from paraxial mesodermal layer.

8. Which lining cells are not derived from endoderm-generated cells? a. Trachea b. Liver c. Pancreas d. Rectum Answer: d. All of the above organs are lined by cells that come from the endoderm except for the rectum, which is lined by cells coming from the ectoderm.

9. What is the best definition of stable transfection?

a. DNA is incorporated into the genome of an animal cell. b. A plasmid is attached to a vector such as a virus c. Artificial DNA is free-floating in the cytoplasm of an animal cell. d. An organism is infected with a bacterium containing foreign DNA. Answer: a. Stable transfection involves the incorporation of foreign DNA into the genome of an animal cell.

10. What is the biggest difference between the DNA microarray technique and the DNA macroarray technique? a. The DNA macroarray technique is faster b. The DNA macroarray technique can sample large samples of DNA at a time c. The DNA microarray technique does not use radiolabeled samples d. There is no difference between the techniques Answer: b. The DNA macroarray technique can sample up to ten thousand samples of DNA at a time but the basic processes involved in doing these two procedures is the same.