Unit 1: An Overview of Cells and Cell Research PDF

Cell Biology Unit I Introduction Origin and Evolution of cells Origin of Eukaryotes and Development of multicellular organisms Cells as experimental models Tools of Cell Biology Molecular composition of cells Cell membrane Prepared by...

Cell Biology Unit I Introduction Origin and Evolution of cells Origin of Eukaryotes and Development of multicellular organisms Cells as experimental models Tools of Cell Biology Molecular composition of cells Cell membrane Prepared by Dr. S. Sujatha  Introduction Cell and molecular biology: An active area of research that is fundamental to all of the biological sciences – growing number of applications in medicine, agriculture, biotechnology, and biomedical engineering – development of new drugs specifically targeted to interfere with the growth of cancer cells and the potential use of stem cells to replace damaged tissues and treat patients suffering from conditions like diabetes, Parkinson's disease, Alzheimer's disease, spinal cord injuries, and heart disease  The Origin and History of Life on Earth What is Life? – Life is, to the best of our knowledge to date, unique to our planet Earth. – There is no simple definition of Life, except that life forms are able to act on their own behalf to support their own existence, and to reproduce themselves. – The field of science known as Biology is dedicated to the study of life, its component species, and the variations within species of life forms.  Earth’s Biosphere and Life Science Earth’s original atmosphere contained little Evolution of oxygen content of or no molecular oxygen, which is required earth’s atmosphere by current animal and advanced plant life forms. The O2 currently in our atmosphere is produced by green plant photosynthesis, which began with reduction of CO2 by anaerobic bacteria, also known as cyanobacteria or “blue-green algae”, inhabiting the oceans in Earth’s early history. Animal life, as we know it (which requires atmospheric oxygen), did not come into existence until about 600 million years ago. An important topic of the present day is that humans are returning CO2 to the atmosphere at a faster rate than plants can reduce it to form O2, which can cause global warming.  Origin and Evolution of cells Cells are divided into two main classes, initially defined by whether they contain a nucleus. Prokaryotic cells (bacteria) lack a nuclear envelope; eukaryotic cells have a nucleus in which the genetic material is separated from the cytoplasm. Prokaryotic cells are generally smaller and simpler than eukaryotic cells; in addition to the absence of a nucleus, their genomes are less complex and they do not contain cytoplasmic organelles or a cytoskeleton. In spite of these differences, the same basic molecular mechanisms govern the lives of both prokaryotes and eukaryotes, indicating that all present-day cells are descended from a single primordial ancestor. Characteristic features Prokaryote Eukaryote Nucleus Absent Present Diameter of a typical cell =1 μm 10-100 μm Cytoskeleton Absent Present Cytoplasmic organelles Absent Present DNA content (base pairs) 1 x 106 to 5 x 106 1.5 x 107 to 5 x 109 Chromosomes Single circular Multiple linear DNA molecule DNA molecules  The First Cell How life originated and how the first cell came into being are matters of speculation, since these events cannot be reproduced in the laboratory. Nonetheless, several types of experiments provide important evidence bearing on some steps of the process. The spontaneous formation of organic molecules was first demonstrated experimentally in the 1950s, when Stanley Miller (then a graduate student) showed that the discharge of electric sparks into a mixture of H2, CH4, Spontaneous formation of organic molecules Water and NH3, in the presence of water, vapor was refluxed through an atmosphere consisting leads to the formation of a variety of CH4, NH3, and H2, into which electric sparks were of organic molecules, including discharged. Analysis of the reaction products revealed several amino acids the formation of a variety of organic molecules, including the amino acids alanine, aspartic acid, glutamic acid, and glycine.  The next step in evolution was the formation of macromolecules. – The monomeric building blocks of macromolecules have been demonstrated to polymerize spontaneously under plausible prebiotic conditions. – But the critical characteristic of the macromolecule from which life evolved must have been the ability to replicate itself. Only a macromolecule capable of directing the synthesis of new copies of itself would have been capable of reproduction and further evolution. – Of the two major classes of informational macromolecules in present-day cells (nucleic acids and proteins), only the nucleic acids are capable of directing their own self-replication. – Nucleic acids can serve as templates for their own synthesis as a result of specific base pairing between complementary nucleotides A critical step in understanding molecular evolution was thus reached in the early 1980s, when it was discovered in the laboratories of Sid Altman and Tom Cech that RNA is capable of catalyzing a number of chemical reactions, including the polymerization of nucleotides. RNA is generally believed to have been the initial genetic system, and an early stage of chemical evolution is thought to have been based on self-replicating RNA molecules—a period of evolution known as the RNA world. Hypothesis: Chemicals to Living cells  The first cell is presumed to have arisen by the enclosure of self-replicating RNA in a membrane composed of phospholipids. Phospholipids are the basic components of all present-day biological membranes, including the plasma membranes of both prokaryotic and eukaryotic cells. When placed in water, phospholipids spontaneously aggregate into a bilayer with their phosphate-containing head groups on the outside in contact with water and their hydrocarbon tails in the interior in contact with each other. Such a phospholipid bilayer forms a stable barrier between two aqueous compartments—for example, separating the interior of the cell from its external environment. The enclosure of self-replicating RNA and associated molecules in a phospholipid membrane would thus have maintained them as a unit, capable of self-reproduction and further evolution. RNA-directed protein synthesis may already have evolved by this time, in which case the first cell would have consisted of self-replicating RNA and its encoded proteins.  Present-day Prokaryotes It includes all the various types of bacteria. Divided into two groups—the archaebacteria and the eubacteria—which diverged early in evolution. Some archaebacteria live in extreme environments, which are unusual today but may have been prevalent in primitive Earth. For example, thermoacidophiles live in hot sulfur springs with temperatures as high as 80°C and pH values as low as 2. The eubacteria include the common forms of present-day bacteria—a large group of organisms that live in a wide range of environments, including soil, water, and other organisms (e.g., human pathogens). Most bacterial cells are spherical, rod-shaped, or spiral, with diameters of 1 to 10 μm. Their DNA contents Bacteria on the point of a pin range from about 0.6 million to 5 million base pairs, an amount sufficient to encode about 5000 different proteins. The largest and most complex prokaryotes are the cyanobacteria, bacteria in which photosynthesis evolved.  Fossil: Prokaryotic cells Modern stromatolites at Shark Bay, Australia. 2.5 billion years ago, the oxygen-producing, non-cyclic pathway of photosynthesis began evolving in cyanobacteria (stromatolites) Oxygen accumulation in the air and seas halted spontaneous formation of molecules of life, formed a protective ozone layer, and spurred evolution of organisms using aerobic respiration  The structure of a typical prokaryotic cell is illustrated by Escherichia coli (E. coli), a common inhabitant of the human intestinal tract. The cell is rod-shaped, about 1 μm in diameter and about 2 μm long. Like most other prokaryotes, E. coli is surrounded by a rigid cell wall composed of polysaccharides and peptides. Beneath the cell wall is the plasma membrane, which is a bilayer of phospholipids and associated proteins. Whereas the cell wall is porous and readily penetrated by a variety of molecules, the plasma membrane provides the functional separation between the inside of the cell and its external environment. The DNA of E. coli is a single circular molecule in the nucleoid, which, in contrast to the nucleus of eukaryotes, is not surrounded by a membrane separating it from the cytoplasm. The cytoplasm contains approximately 30,000 ribosomes (the sites of protein synthesis), which account for its granular appearance.  Eukaryotic cells All eukaryotic cells are surrounded by a plasma membrane and contain ribosomes. Eukaryotic cells are much more complex than prokaryotes and contain a nucleus, a variety of cytoplasmic organelles, and a cytoskeleton. The largest and most prominent organelle of eukaryotic cells is the nucleus, with a diameter of approximately 5 μm. – The nucleus contains the genetic information of the cell, which in eukaryotes is organized as linear rather than circular DNA molecules. – The nucleus is the site of DNA replication and of RNA synthesis; the translation of RNA into proteins takes place on ribosomes in the cytoplasm. In addition to a nucleus, eukaryotic cells contain a variety of membrane- enclosed organelles within their cytoplasm. These organelles provide compartments in which different metabolic activities are localized. Two of these organelles, mitochondria and chloroplasts, play critical roles in energy metabolism. Mitochondria, which are found in almost all eukaryotic cells, are the sites of oxidative metabolism and are thus responsible for generating most of the ATP derived from the breakdown of organic molecules.  Chloroplasts are the sites of photosynthesis and are found only in the cells of plants and green algae. Lysosomes and peroxisomes also provide specialized metabolic compartments for the digestion of macromolecules and for various oxidative reactions, respectively. In addition, most plant cells contain large vacuoles that perform a variety of functions, including the digestion of macromolecules and the storage of both waste products and nutrients. Two cytoplasmic organelles, the endoplasmic reticulum and the Golgi apparatus, are specifically devoted to the sorting and transport of proteins destined for secretion, incorporation into the plasma membrane, and incorporation into lysosomes. The endoplasmic reticulum is an extensive network of intracellular membranes, extending from the nuclear membrane throughout the cytoplasm. It functions not only in the processing and transport of proteins, but also in the synthesis of lipids. From the endoplasmic reticulum, proteins are transported within small membrane vesicles to the Golgi apparatus, where they are further processed and sorted for transport to their final destinations. In addition to this role in protein transport, the Golgi apparatus serves as a site of lipid synthesis and (in plant cells) as the site of synthesis of some of the polysaccharides that compose the cell wall. Eukaryotic cells have another level of internal organization: the cytoskeleton, a network of protein filaments extending throughout the cytoplasm. The cytoskeleton provides the structural framework of the cell, determining cell shape and the general organization of the cytoplasm. Also, responsible for movement and transport  The Origin of Eukaryotes A critical step in the evolution of eukaryotic cells was the acquisition of membrane-enclosed subcellular organelles The organelles of eukaryotes are thought to have arisen by endosymbiosis—one cell living inside another. In particular, eukaryotic organelles are thought to have evolved from prokaryotic cells living inside the ancestors of eukaryotes. The hypothesis that eukaryotic cells evolved by endosymbiosis is particularly well supported by studies of mitochondria and chloroplasts, which are thought to have evolved from eubacteria living in larger cells. Evidence for Endosymbiosis  Both mitochondria and chloroplasts are similar to bacteria in size, and like bacteria, they reproduce by dividing in two. – Most important, both mitochondria and chloroplasts contain their own DNA, which encodes some of their components. – The mitochondrial and chloroplast DNAs are replicated each time the organelle divides, and the genes they encode are transcribed within the organelle and translated on organelle ribosomes. – Mitochondria and chloroplasts thus contain their own genetic systems, which are distinct from the nuclear genome of the cell. Furthermore, the ribosomes and ribosomal RNAs of these organelles are more closely related to those of bacteria than to those encoded by the nuclear genomes of eukaryotes. An endosymbiotic origin for these organelles is now generally accepted, with mitochondria thought to have evolved from aerobic eubacteria and chloroplasts from photosynthetic eubacteria, such as the cyanobacteria.  A recent hypothesis explains the mosaic nature of eukaryotic genomes by proposing that the genome of eukaryotes arose from a fusion of archaebacterial and eubacterial genomes. According to this proposal, an endosymbiotic association between a eubacterium and an archaebacterium was followed by fusion of the two prokaryotic genomes, giving rise to an ancestral eukaryotic genome with contributions from both eubacteria and archaebacteria. The simplest version of this hypothesis is that an initial endosymbiotic relationship of a eubacterium living inside an archaebacterium gave rise not only to mitochondria but also to the genome of eukaryotic cells, containing genes derived from both prokaryotic ancestors.  Membrane invagination theory: ▪ Plasma membrane of prokaryotic cells folded in on itself forming pockets thereby isolating metabolic reactions. ▪ Pockets pinched off partitioning function into membrane enclosed organelles. ▪ These organelles increased in complexity and specialization forming complex organelles found in eukaryotic cells. Secondary Endosymbiosis: ▪ Refers to process wherein the eukaryotes engulfs another eukaryotes. ▪ Chloroplasts of algae have been derived by engulfing the photosynthetic eukaryotes. ▪ Eukaryotic nature of the endosymbiont can be seen by its retention of vestige of nucleus called nucleomorph. ▪ In 2005 Okamoto and Inouye discovered a heterotrophic flagellate that engulfs unicellular alga. ▪ Once inside, the alga loses flagella and cytoskeleton while the host switches from heterotrophic nutrition to autotrophic nutrition. ▪ The host divides by mitosis. One daughter cell gets the plastid while the other daughter cells regrows its feeding apparatus and is ready to engulf another alga.  Development of Multicellular Organisms Many eukaryotes are unicellular organisms that, like bacteria, consist of only single cells capable of self-replication. The simplest eukaryotes are the yeasts (S. cerevisiae). Yeasts are more complex than bacteria, but much smaller and simpler than the cells of animals or plants. Other unicellular eukaryotes, however, are far more complex cells, some containing as much DNA as human cells have. They include organisms specialized to perform a variety of tasks, including photosynthesis, movement, and the capture and ingestion of other organisms as food. Amoeba proteus, for example, is a large, complex cell. Its volume is more than 100,000 times that of E. coli, and its length can exceed 1 mm when the cell is fully extended Other unicellular eukaryotes (the green algae) contain chloroplasts and are able to carry out photosynthesis.  Multicellular organisms evolved from unicellular eukaryotes more than 1 billion years ago. Some unicellular eukaryotes form multicellular aggregates that appear to represent an evolutionary transition from single cells to multicellular organisms. For instance, the cells of many algae (e.g., the green alga Volvox) associate with each other to form multicellular colonies, which are thought to have been the evolutionary precursors of present-day plants.  Plants are composed of fewer cell types than are animals, but each different kind of plant cell is specialized to perform specific tasks required by the organism as a whole. The cells of plants are organized into three main tissue systems: ground tissue, dermal tissue, and vascular tissue. The cells found in animals are considerably more diverse than those of plants.  The human body, for example, is composed of more than 200 different kinds of cells, which are generally considered to be components of five main types of tissues: epithelial tissue, connective tissue, blood, nervous tissue, and muscle. The evolution of animals clearly involved the development of considerable diversity and specialization at the cellular level. Understanding the mechanisms that control the growth and differentiation of such a complex array of specialized cells, starting from a single fertilized egg, is one of the major challenges facing contemporary cell and molecular biology.  Cells as Experimental Models The evolution of present-day cells from a common ancestor has important implications for cell and molecular biology as an experimental science. Several different kinds of cells and organisms are commonly used as experimental models to study various aspects of cell and molecular biology. The features of some of these cells that make them particularly advantageous as experimental models because of the availability of complete genome sequences  Escherichia coli Because of their comparative simplicity, prokaryotic cells (bacteria) are ideal models for studying many fundamental aspects of biochemistry and molecular biology. Most of our present concepts of molecular biology—including our understanding of DNA replication, the genetic code, gene expression, and protein synthesis—derive from studies of this humble bacterium. Under optimal culture conditions, E. coli divide every 20 minutes. Because bacterial colonies containing as many as 108 cells can develop overnight, selecting genetic variants of an E. coli strain—for example, mutants that are resistant to an antibiotic, such as penicillin—is easy and rapid. The ability of E. coli to carry out these biosynthetic reactions (amino acids and nucleotides) in simple defined media has made them extremely useful in elucidating the biochemical pathways involved.  Yeast Yeasts have provided a crucial model for studies of many fundamental aspects of eukaryotic cell biology. The genome of the most frequently studied yeast, Saccharomyces cerevisiae, consists of 12 million base pairs of DNA and contains about 6000 genes. It contains a distinct nucleus surrounded by a nuclear membrane, its genomic DNA is organized as 16 linear chromosomes, and its cytoplasm contains a cytoskeleton and subcellular organelles. Yeasts do not replicate as rapidly as bacteria but still divide as frequently as every 2 hours and they can easily be grown as colonies from a single cell. Yeast mutants have been important in understanding DNA replication, transcription, RNA processing, protein sorting, and the regulation of cell division. General principles of cell structure and function were revealed by studies of yeasts  Caenorhabditis elegans The nematode Caenorhabditis elegans is one of the most widely used models for studies of animal development and cell differentiation. The genome of C. elegans contains approximately 19,000 genes. Adult worms consist of only 959 somatic cells, plus 1000 to 2000 germ cells. C. elegans can be easily grown and subjected to genetic manipulations in the laboratory. Genetic studies have also identified many of the mutations responsible for developmental abnormalities, leading to the isolation and characterization of critical genes that control nematode development and differentiation. Similar genes have also been found to function in complex animals (including humans), making C. elegans an important model for studies of animal development.  Drosophila melanogaster The fruit fly, Drosophila melanogaster, has been a crucial model organism in developmental biology. The genome of Drosophila is 180 million base pairs, larger than that of C. elegans, but the Drosophila genome only contains about 14,000 genes. Drosophila can be easily maintained and bred in the laboratory, and the short reproductive cycle of Drosophila (about 2 weeks) makes it an useful organism for genetic experiments. Many fundamental concepts of genetics—such as the relationship between genes and chromosomes—were derived from studies of Drosophila early in the twentieth century Studies of Drosophila have led to advances in understanding the molecular mechanisms of animal development, particularly, formation of the body plan of complex multicellular organisms.  Arabidopsis thaliana The small flowering plant, Arabidopsis thaliana, is widely used as a model to study the molecular biology of plants. Arabidopsis is notable for its genome of only about 125 million base pairs. Although Arabidopsis contains a total of about 26,000 genes, many of these are repeated, so the number of unique genes in Arabidopsis is approximately 15,000—a complexity similar to that of C. elegans and Drosophila. Arabidopsis is relatively easy to grow in the laboratory Studies have led to the identification of genes involved in plant development, such as the development of flowers. Analysis of these genes have revealed similarities and also, differences, between the mechanisms that control the development of plants and animals.  Vertebrates The human genome is approximately 3 billion base pairs—about 20-30 times larger than the genomes of C. elegans, Drosophila, or Arabidopsis - and contains 20,000 to 25,000 genes. The human body is composed of more than 200 different kinds of specialized cell types. This complexity makes the vertebrates difficult to study from the standpoint of cell and molecular biology Moreover, an understanding of many questions of immediate practical importance (e.g., in medicine) must be based directly on studies of human (or closely related) cell types. One important approach to studying human and other mammalian cells is to grow isolated cells in culture, where they can be manipulated under controlled laboratory conditions. The use of cultured cells has allowed studies of many aspects of mammalian cell biology, including experiments that have elucidated the mechanisms of DNA replication, gene expression, protein synthesis and processing, cell division, and cell signaling mechanisms that normally control cell growth and differentiation  In humans, nerve cell axons may be more than a meter long, and some invertebrates, such as the squid, have giant neurons with axons as large as 1 mm in diameter. – Because of their highly specialized structure and function, these giant neurons have provided important models for studies of ion transport across the plasma membrane, and of the role of the cytoskeleton in the transport of cytoplasmic organelles. The frog, Xenopus laevis, is an important model for studies of early vertebrate development, differentiation, and embryonic cell division. The zebra fish possesses a number of advantages for genetic studies of vertebrate development. – These small fish are easy to maintain in the laboratory and they reproduce rapidly – The embryos develop outside of the mother and are transparent, so that early stages of development can be easily observed.  Among mammals, the mouse is the most suitable for genetic analysis, which is facilitated by the availability of its complete genome sequence. Recent advances in molecular biology have enabled the production of genetically engineered mice in which specific mutant genes have been introduced into the mouse germ line, allowing the functions of these genes to be studied in the context of the whole animal. The suitability of the mouse as a model for human development is indicated not only by the similarity of the mouse and human genomes but also by the fact that mutations in homologous genes result in similar developmental defects in both species; piebaldism is a striking example.  Tools of Cell Biology As in all experimental sciences, research in cell biology depends on the laboratory methods that can be used to study cell structure and function. Many important advances in understanding cells have directly followed the development of new methods that have opened novel avenues of investigation. An appreciation of the experimental tools available to the cell biologist is thus critical to understanding both the current status and future directions of this rapidly moving area of science. Some of the important general methods of cell biology are described in the sections that follow.  Microscopes are a Necessary tool of Cell Biology The light microscope remains a basic tool of cell biologists and can to magnify objects up to about a thousand times.  Microscopy 10 m Human height Scientists use microscopes to visualize 1m Length of some nerve and muscle cells too small to see with the naked Unaided eye cells 0.1 m eye Chicken egg In a light microscope (LM), visible 1 cm light passes through a specimen and Frog egg then through glass lenses, which 1 mm magnify the image Light microscope 100 µm The quality of an image depends on Most plant and animal cells – Magnification, the ratio of an 10 µm Nucleus object’s image size to its real size Most bacteria Electron microscope Mitochondrion 1 µm – Resolution, the measure of the clarity of the image, or the 100 nm Smallest bacteria Viruses minimum distance of two Ribosomes distinguishable points 10 nm Proteins – Contrast, visible differences in Lipids 1 nm parts of the sample Small molecules 0.1 nm Atoms  LMs can magnify effectively to about 1,000 times the size of the actual specimen Various techniques enhance contrast and enable cell components to be stained or labeled Most subcellular structures, including organelles (membrane-enclosed compartments), are too small to be resolved by an LM Bright-field microscopy, in which light passes directly through the cell, is routinely used to study various aspects of cell structure because of its simplicity. Brightfield micrograph of a stained section of benign kidney tumor.  Phase-contrast microscopy and differential interference-contrast microscopy use optical systems that convert variations in density or thickness between different parts of the cell to differences in contrast that can be seen in the final image. FIGURE 1.24 Microscopic observation of living cells Photomicrographs of human cheek cells obtained with (A) bright-field, (B) phase-contrast, and (C) differential interference-contrast microscopy. (Courtesy of Mort Abramowitz,  Flourescence Microscopy Fluorescence microscopy is a widely used and very sensitive method for studying the intracellular distribution of molecules. – The green fluorescent protein (GFP) of jellyfish is used to visualize proteins within living cells. – Fluorescence recovery after photobleaching (FRAP) is used to study the movements of GFP-labeled proteins.  A microtubule associated protein fused to GFP (green flourescent protein) was introduced into mouse neurons in cell culture. The nuclei of the cells is stained blue. Allows for the determination of cellular localization. FIGURE 1.27 Fluorescence microscopy of a protein labeled with GFP A microtubule-associated protein fused to GFP was introduced into mouse neurons in culture and visualized by fluorescence microscopy. Nuclei are stained blue. (From A. Cariboni, 2004. Nature Cell Biol. 6:929.)  Confocal microscopy Confocal microscopy allows images of increased contrast and detail to be obtained by analyzing fluorescence from only a single point in the specimen. A small point of light, usually supplied by a laser, is focused on the specimen at a particular depth. The emitted fluorescent light is then collected using a detector, such as a video camera. Before the emitted light reaches the detector, however, it must pass through a pinhole aperture (called a confocal aperture) placed at precisely the point where light emitted from the chosen depth of the specimen comes to a focus. Consequently, only light emitted from the plane of focus is able to reach the detector. Scanning across the specimen generates a two-dimensional image of the plane of focus, a much sharper image than that obtained with standard fluorescence microscopy. Moreover, a series of images obtained at different depths can be used to reconstruct a three- dimensional image of the sample.  Electron Microscopy The electron microscope was developed jointly by Albert Claude, Keith Porter, and George Palade in the 1940s and 1950s. The electron microscope can achieve much greater resolution than that obtained with the light microscope. FIGURE 1.33 Positive staining Transmission electron micrograph of a positively stained white blood cell. (Don W. Fawcett/Visuals Unlimited.)  (a) Scanning electron microscopy (SEM) Two basic types of electron 1 µm Cilia microscopes (EMs) are used to study subcellular structures Scanning electron microscopes (SEMs) focus a beam of electrons onto the surface of a specimen, providing images that look 3-D Longitudinal Cross section Transmission electron section of of cilium 1 µm cilium microscopes (TEMs) focus a beam of electrons through a specimen TEMs are used mainly to study the internal structure of cells (b) Transmission electron  Subcellular fractionation Cell fractionation takes cells apart and separates the major organelles from one another Differential centrifugation separates and isolates eukaryotic cell organelles on the basis of their size and density for use in biochemical studies. The force of an ultracentrifuge causes cell components to move toward the bottom of the centrifuge tube and form a pellet at a rate that depends on their size and density. Cell fractionation enables scientists to determine the functions of organelles Biochemistry and cytology help correlate cell function with structure  In velocity centrifugation, the starting material is layered on top of the sucrose gradient. Particles of different sizes sediment through the gradient at different rates, moving as discrete bands. Following centrifugation, the collection of individual fractions of the gradient provides sufficient resolution to separate organelles of similar size, such as mitochondria, lysosomes, and peroxisomes.  Growth of Animal Cells in Culture In vitro cell culture systems enable scientists to: – study cell growth and differentiation – perform genetic manipulations to understand gene structure and function. Culture media contains: – Serum – Salts – Glucose – Various amino acids and vitamins that the cells do not make for themselves.  Primary cultures are the original cultures established from a tissue. Permanent (or immortal) cell lines are embryonic stem cells or tumor cells that proliferate indefinitely in culture.  Culture of Plant Cells Plant cells can also be cultured in nutrient media containing appropriate growth regulatory molecules. In contrast to the polypeptide growth factors that regulate the proliferation of most animal cells, the growth regulators of plant cells are small molecules that can pass through the plant cell wall. When provided with appropriate mixtures of these growth regulatory molecules, many types of plant cells proliferate in culture, producing a mass of undifferentiated cells called a callus It is noteworthy that many plant cells are capable of forming any of the different cell types and tissues ultimately needed to regenerate an entire plant. In many cases, even an entire plant can be regenerated from a single cultured cell. In addition to its theoretical interest, the ability to produce a new plant from a single cell that has been manipulated in culture makes it easy to introduce genetic alterations into plants, opening important possibilities for agricultural genetic engineering.  Molecular Composition of Cells Cells are composed of water, inorganic ions, and carbon-containing (organic) molecules. Water is the most abundant molecule in cells, accounting for 70% or more of total cell mass. The critical property of water in this respect is that it is a polar molecule, in which the hydrogen atoms have a slight positive charge and the oxygen has a slight negative charge. – Because of their polar nature, water molecules can form hydrogen bonds with each other or with other polar molecules, as well as interact with positively or negatively charged ions. – As a result of these interactions, ions and polar molecules are readily soluble in water (hydrophilic). In contrast, nonpolar molecules, which cannot interact with water, are poorly soluble in an aqueous environment (hydrophobic). – Consequently, nonpolar molecules tend to minimize their contact with water by associating closely with each other instead. Such interactions of polar and nonpolar molecules with water and with each other play crucial roles in the formation of biological structures, such as cell membranes. The inorganic ions of the cell, including sodium (Na+), potassium (K+), magnesium (Mg2+), calcium (Ca2+), phosphate (HPO42-), chloride (Cl-), and bicarbonate (HCO3-), constitute 1% or less of the cell mass. These ions are involved in a number of aspects of cell metabolism, and thus play critical roles in cell function.  The organic molecules are the unique constituents of cells. Most of these organic compounds belong to one of four classes of molecules: carbohydrates, lipids, proteins, and nucleic acids. Proteins, nucleic acids, and most carbohydrates (the polysaccharides) are macromolecules formed by the joining (polymerization) of hundreds or thousands of low molecular-weight precursors: amino acids, nucleotides, and simple sugars, respectively. Such macromolecules constitute 80 to 90% of the dry weight of most cells. Lipids are the other major constituent of cells. The remainder of the cell mass is composed of a variety of small organic molecules, including macromolecular precursors.  Carbohydrates The carbohydrates include simple sugars as well as polysaccharides. These simple sugars, such as glucose, are the major nutrients of cells. – Breakdown of these sugars provides both a source of cellular energy and the starting material for the synthesis of other cell constituents. Polysaccharides are storage forms of sugars and form structural components of the cell. – In addition, polysaccharides and shorter polymers of sugars act as markers for a variety of cell recognition processes, including the adhesion of cells to their neighbors and the transport of proteins to appropriate intracellular destinations.  The basic formula for monosaccharides is (CH2O)n from which the name carbohydrate is derived (C = "carbo" and H2O = "hydrate"). The six-carbon (n = 6) sugar glucose (C6H12O6) is especially important in cells, since it provides the principal source of cellular energy. The cyclized sugars exist in two alternative forms (called α or β), depending on the configuration of carbon 1.  Monosaccharides can be joined together by dehydration reactions in which H20 is removed and the sugars are linked by a glycosidic bond between two of their carbons. If only a few sugars are joined together, the resulting polymer is called an oligosaccharide. If a large number (hundreds or thousands) of sugars are involved, the resulting polymers are macromolecules called polysaccharides.  Two common polysaccharides—glycogen and starch—are the storage forms of carbohydrates in animal and plant cells, respectively. – composed entirely of glucose molecules in the α configuration. Cellulose is the principal structural component of the plant cell wall. – composed entirely of glucose molecules.in the β configuration – It is an unbranched polysaccharide. Oligosaccharides and polysaccharides are important for energy storage , cell structure, and informational processes. – Glycoproteins play important roles in protein folding and serve as markers to target proteins for transport to the cell surface or incorporation into different subcellular organelles. – serve as markers on the surface of cells, playing important roles in cell recognition and the interactions between cells in tissues of multicellular organisms.  Lipids Lipids have three major roles in cells. – First, they provide an important form of energy storage. – Second, and of great importance in cell biology, lipids are the major components of cell membranes. – Third, lipids play important roles in cell signaling, both as steroid hormones (e.g., estrogen and testosterone) and as messenger molecules that convey signals from cell surface receptors to targets within the cell. The simplest lipids are fatty acids, which consist of long hydrocarbon chains, most frequently containing 16 or 18 carbon atoms, with a carboxyl group (COO-) at one end. – Unsaturated fatty acids contain one or more double bonds between carbon atoms – In Saturated fatty acids, all of the carbon atoms are bonded to the maximum number of hydrogen atoms. The long hydrocarbon chains of fatty acids contain only nonpolar C—H bonds, which are unable to interact with water. The hydrophobic nature of these fatty acid chains is responsible for much of the behavior of complex lipids, particularly in the formation of biological membranes.  Fatty acids are stored in the form of triacylglycerols, or fats, which consist of three fatty acids linked to a glycerol molecule. Triacylglycerols are insoluble in water and therefore accumulate as fat droplets in the cytoplasm. – When required, they can be broken down for use in energy-yielding reactions. Fats are a more efficient form of energy storage than carbohydrates, yielding more than twice as much energy per weight of material broken down.  Phospholipids, the principal components of cell membranes, consist of two fatty acids joined to a polar head group. All phospholipids have hydrophobic tails, consisting of the two hydrocarbon chains, and hydrophilic head groups, consisting of the phosphate group and its polar Figure 1: The lipid bilayer and the structure and composition of a attachments. glycerophospholipid molecule – Consequently, phospholipids (A) The plasma membrane of a cell is a bilayer of glycerophospholipid are amphipathic molecules, molecules. (B) A single glycerophospholipid molecule is composed of part water-soluble and part two major regions: a hydrophilic head (green) and hydrophobic tails water-insoluble. (purple). (C) The subregions of a glycerophospholipid molecule; phosphatidylcholine is shown as an example. The hydrophilic head is This property of composed of a choline structure (blue) and a phosphate (orange). phospholipids is the basis for This head is connected to a glycerol (green) with two hydrophobic tails (purple) called fatty acids. (D) This view shows the specific atoms the formation of biological within the various subregions of the phosphatidylcholine molecule. membranes. Note that a double bond between two of the carbon atoms in one of the hydrocarbon (fatty acid) tails causes a slight kink on this molecule, so it appears bent.  In addition to phospholipids, many cell membranes contain glycolipids and cholesterol. Glycolipids consist of two hydrocarbon chains linked to polar head groups that contain carbohydrates. They are thus similar to the phospholipids in their general organization as amphipathic molecules. Cholesterol, in contrast, consists of four hydrocarbon rings rather than linear hydrocarbon chains. The hydrocarbon rings are strongly hydrophobic, but the hydroxyl (OH) group attached to one end of cholesterol is weakly hydrophilic, so cholesterol is also amphipathic.  Nucleic Acids The nucleic acids—DNA and RNA—are the principal informational molecules of the cell. Deoxyribonucleic acid (DNA) has a unique role as the genetic material, which in eukaryotic cells is located in the nucleus. Different types of ribonucleic acid (RNA) participate in a number of cellular activities. – Messenger RNA (mRNA) carries information from DNA to the ribosomes, where it serves as a template for protein synthesis. – Two other types of RNA (ribosomal RNA and transfer RNA) are involved in protein synthesis. – Still other kinds of RNAs are involved in the processing and transport of both RNAs and proteins. DNA and RNA are polymers of nucleotides, which consist of purine and pyrimidine bases linked to phosphorylated sugars. DNA contains two purines (adenine and guanine) and two pyrimidines (cytosine and thymine). Adenine, guanine, and cytosine are also present in RNA, but RNA contains uracil in place of thymine. The bases are linked to sugars (2'-deoxyribose in DNA, or ribose in RNA) to form nucleosides. Nucleotides additionally contain one or more phosphate groups linked to the 5' carbon of nucleoside sugars.  DNA is a double-stranded molecule consisting of two polynucleotide chains running in opposite directions – The bases are on the inside of the molecule, and the two chains are joined by hydrogen bonds between complementary base pairs—adenine pairing with thymine and guanine with cytosine The important consequence of such complementary base pairing is that one strand of DNA (or RNA) can act as a template to direct the synthesis of a complementary strand. Nucleic acids are thus uniquely capable of directing their own self-replication, allowing them to function as the fundamental informational molecules of the cell. The information carried by DNA and RNA directs the synthesis of specific proteins, which control most cellular activities. Nucleotides are not only important as the building blocks of nucleic acids; they also play critical roles in other cell processes. – Perhaps the most prominent example is adenosine 5'-triphosphate (ATP), which is the principal form of chemical energy within cells. – Other nucleotides similarly function as carriers of either energy or reactive chemical groups in a wide variety of metabolic reactions. – In addition, some nucleotides (e.g., cyclic AMP) are important signaling molecules within cells  Proteins Proteins (Greek word) = “proteios,” meaning "of the first rank." The primary responsibility of proteins is to execute the tasks directed by the genetic information of the nucleic acids. Proteins are the most diverse of all macromolecules, and each cell contains several thousand different proteins, which perform a wide variety of functions. – serving as structural components of cells and tissues, – acting in the transport and storage of small molecules (e.g., the transport of oxygen by hemoglobin), – transmitting information between cells (e.g., protein hormones), and providing a defense against infection (e.g., antibodies). The most fundamental property of proteins, however, is their ability to act as enzymes, catalyze nearly all the chemical reactions in biological systems.  Proteins are polymers of 20 different amino acids. Each amino acid consists of a carbon atom (called the α carbon) bonded to a carboxyl group (COO-), an amino group (NH3+), a hydrogen atom, and a distinctive side chain). The specific chemical properties of the different amino acid side chains determine the roles of each amino acid in protein structure and function. Amino acids are joined together by peptide bonds between the a amino group of one amino acid and the a carboxyl group of a second Polypeptides are linear chains of amino acids, usually hundreds or thousands of amino acids in length. – Each polypeptide chain has two distinct ends, one terminating in an a amino group (the amino, or N, terminus) and the other in an a carboxyl group (the carboxy, or C, terminus). – Polypeptides are synthesized from the amino to the carboxy terminus, and the sequence of amino acids in a polypeptide is written (by convention) in the same order.  Protein structure is generally described as having four levels. The primary structure of a protein is the sequence of amino acids in its polypeptide chain. The secondary structure is the regular arrangement of amino acids within localized regions of the polypeptide. Two types of secondary structure, (first proposed by Linus Pauling and Robert Corey in 1951), the α helix and the β sheet. – Both of these secondary structures are held together by hydrogen bonds between the CO and NH groups of peptide bonds. – An α helix is formed when a region of a polypeptide chain coils around itself, with the CO group of one peptide bond forming a hydrogen bond with the NH group of a peptide bond located four residues downstream in the linear polypeptide chain – In contrast, a β sheet is formed when two parts of a polypeptide chain lie side by side with hydrogen bonds between them. Such β sheets can be formed between several polypeptide strands, which can be oriented either parallel or antiparallel to each other.  Tertiary structure is the folding of the polypeptide chain as a result of interactions between the side chains of amino acids that lie in different regions of the primary sequence In most proteins, combinations of α helices and β sheets, connected by loop regions of the polypeptide chain, fold into compact globular structures called domains, which are the basic units of tertiary structure. – Small proteins, such as ribonuclease or myoglobin, contain only a single domain; larger proteins may contain a number of different domains, which are frequently associated with distinct functions. – A critical determinant of tertiary structure is the localization of hydrophobic amino acids in the interior of the protein and of hydrophilic amino acids on the surface, where they interact with water.  The fourth level of protein structure, quaternary structure, consists of the interactions between different polypeptide chains in proteins composed of more than one polypeptide. – Hemoglobin is composed of four polypeptide chains held together by the same types of interactions that maintain tertiary structure  Cell Membranes The structure and function of cells are critically dependent on membranes, which not only separate the interior of the cell from its environment but also define the internal compartments of eukaryotic cells, including the nucleus and cytoplasmic organelles. The formation of biological membranes is based on the properties of lipids, and all cell membranes share a common structural organization: bilayers of phospholipids with associated proteins. These membrane proteins are responsible for many specialized functions; some act as receptors that allow the cell to respond to external signals, some are responsible for the selective transport of molecules across the membrane, and others participate in electron transport and oxidative phosphorylation. In addition, membrane proteins control the interactions between cells of multicellular organisms.  Membrane Lipids Phospholipids of cell membranes are amphipathic molecules – two hydrophobic fatty acid chains linked to a phosphate-containing hydrophilic head group – Since fatty acid tails are poorly soluble in water, phospholipids spontaneously form bilayers in aqueous solutions, with the hydrophobic tails buried in the interior of the membrane and the polar head groups exposed on both sides, in contact with water – They form a stable barrier between two aqueous compartments  Lipids constitute approximately 50% of the mass of most cell membranes, although this proportion varies depending on the type of membrane. – Plasma membranes, for example, are approximately 50% lipid and 50% protein. – The inner membrane of mitochondria, on the other hand, contains an unusually high fraction (about 75%) of protein, reflecting the abundance of protein complexes involved in electron transport and oxidative phosphorylation. – The plasma membrane of E. coli consists predominantly of phosphatidylethanolamine, which constitutes 80% of total membrane lipid. Mammalian plasma membranes are more complex, containing four major phospholipids— phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and sphingomyelin—which together constitute 50 to 60% of total membrane lipid. In addition to the phospholipids, the plasma membranes of animal cells contain glycolipids and cholesterol, which generally correspond to about 40% of the total membrane lipid molecules.  An important property of lipid bilayers is that they behave as two-dimensional fluids in which individual molecules (both lipids and proteins) are free to rotate and move in lateral directions. Such fluidity is a critical property of membranes and is determined by both temperature and lipid composition. – For example, the interactions between shorter fatty acid chains are weaker than those between longer chains, so membranes containing shorter fatty acid chains are less rigid and remain fluid at lower temperatures. – Lipids containing unsaturated fatty acids similarly increase membrane fluidity because the presence of double bonds introduces kinks in the fatty acid chains, making them more difficult to pack together.  Because of its hydrocarbon ring structure, cholesterol plays a distinct role in determining membrane fluidity. Cholesterol molecules insert into the bilayer with their polar hydroxyl groups close to the hydrophilic head groups of the phospholipids. – The rigid hydrocarbon rings of cholesterol therefore interact with the regions of the fatty acid chains that are adjacent to the phospholipid head groups. – This interaction decreases the mobility of the outer portions of the fatty acid chains, making this part of the membrane more rigid. – On the other hand, insertion of cholesterol interferes with interactions between fatty acid chains, thereby maintaining membrane fluidity at lower temperatures.  Membrane Proteins Proteins are the other major constituent of cell membranes, constituting 25 to 75% of the mass of the various membranes of the cell. The current model of membrane structure, proposed by Jonathan Singer and Garth Nicolson in 1972, views the membrane as a fluid mosaic in which proteins are inserted into a lipid bilayer While phospholipids provide the basic structural organization of membranes, membrane proteins carry out the specific functions of the different membranes of the cell. These proteins are divided into two general classes, based on the nature of their association with the membrane. – Integral membrane proteins (called transmembrane proteins) are embedded directly within the lipid bilayer. – Peripheral membrane proteins are not inserted into the lipid bilayer but are associated with the membrane indirectly, generally by interactions with integral membrane proteins.  Most integral membrane proteins (amphipathic) span the lipid bilayer, with portions exposed on both sides of the membrane. – The membrane-spanning portions of these proteins are usually α-helical regions of 20 to 25 nonpolar amino acids. – The hydrophobic side chains of these amino acids interact with the fatty acid chains of membrane lipids, and the formation of an α helix neutralizes the polar character of the peptide bonds – The only other protein structure known to span lipid bilayers is the β-barrel, formed by the folding of (3 sheets into a barrel-like structure, which is found in some transmembrane proteins of bacteria, chloroplasts, and mitochondria. Some transmembrane proteins span the membrane only once; others have multiple membrane-spanning regions. Most transmembrane proteins of eukaryotic plasma membranes have been modified by the addition of carbohydrates, which are exposed on the surface of the cell and may participate in cell-cell interactions.  Proteins can also be anchored in membranes by lipids that are covalently attached to the polypeptide chain. Distinct lipid modifications anchor proteins to the cytosolic and extracellular faces of the plasma membrane. – Proteins can be anchored to the cytosolic face of the membrane either by the addition of a 14-carbon fatty acid (myristic acid) to their amino terminus or by the addition of either a 16-carbon fatty acid (palmitic acid) or 15- or 20-carbon prenyl groups to the side chains of cysteine residues. – Alternatively, proteins are anchored to the extracellular face of the plasma membrane by the addition of glycolipids to their carboxy terminus.