Inside the cell there is a large fluid-filled space called the cytoplasm, sometimes called the cytosol. In prokaryotes, this space is relatively free of compartments. In eukaryotes, the cytosol is the "soup" within which all of the cell's organelles reside. It is also the home of the cytoskeleton. The cytosol contains dissolved nutrients, helps break down waste products, and moves material around the cell through a process called cytoplasmic streaming. The nucleus often flows with the cytoplasm changing its shape as it moves. The cytoplasm also contains many salts and is an excellent conductor of electricity, creating the perfect environment for the mechanics of the cell. The function of the cytoplasm, and the organelles which reside in it, are critical for a cell's survival.

Two different kinds of genetic material exist: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Most organisms are made of DNA, but a few viruses have RNA as their genetic material. The biological information contained in an organism is encoded in its DNA or RNA sequence.

Prokaryotic genetic material is organized in a simple circular structure that rests in the cytoplasm. Eukaryotic genetic material is more complex and is divided into discrete units called genes. Human genetic material is made up of two distinct components: the nuclear genome and the mitochondrial genome. The nuclear genome is divided into 24 linear DNA molecules, each contained in a different chromosome. The mitochondrial genome is a circular DNA molecule separate from the nuclear DNA. Although the mitochondrial genome is very small, it codes for some very important proteins. Interestingly, as much as 98 percent of human DNA does not code for a specific product.

The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a different function. Cells also have a set of "little organs", called organelles, which are adapted and/or specialized for carrying out one or more vital functions. Organelles are found only in eukaryotes and are always surrounded by a protective membrane. It is important to know some basic facts about the following organelles.

The nucleus is the most conspicuous organelle found in a eukaryotic cell. It houses the cell's chromosomes and is the place where almost all DNA replication and RNA synthesis occur. The nucleus is spheroid in shape and separated from the cytoplasm by a membrane called the nuclear envelope. The nuclear envelope isolates and protects a cell's DNA from various molecules that could accidentally damage its structure or interfere with its processing. During processing, DNA is transcribed, or synthesized, into a special RNA, called mRNA. This mRNA is then transported out of the nucleus, where it is translated into a specific protein molecule. In prokaryotes, DNA processing takes place in the cytoplasm.

Ribosomes are found in both prokaryotes and eukaryotes. The ribosome is a large complex composed of many molecules, including RNAs and proteins, and is responsible for processing the genetic instructions carried by an mRNA. The process of converting an mRNA's genetic code into the exact sequence of amino acids that make up a protein is called translation. Protein synthesis is extremely important to all cells, and therefore a large number of ribosomes—sometimes hundreds or even thousands—can be found throughout a cell.

Ribosomes float freely in the cytoplasm or sometimes bind to another organelle called the endoplasmic reticulum. Ribosomes are composed of one large and one small subunit, each having a different function during protein synthesis.

Mitochondria are self-replicating organelles that occur in various numbers, shapes, and sizes in the cytoplasm of all eukaryotic cells. As mentioned earlier, mitochondria contain their own genome that is separate and distinct from the nuclear genome of a cell. Mitochondria have two functionally distinct membrane systems separated by a space: the outer membrane, which surrounds the whole organelle; and the inner membrane, which is thrown into folds or shelves that project inward. These inward folds are called cristae. The number and shape of cristae in mitochondria differ, depending on the tissue and organism in which they are found, and serve to increase the surface area of the membrane.

Mitochondria play a critical role in generating energy in the eukaryotic cell, and this process involves a number of complex pathways. Let's break down each of these steps so that you can better understand how food and nutrients are turned into energy packets and water. Some of the best energy-supplying foods that we eat contain complex sugars. These complex sugars can be broken down into a less chemically complex sugar molecule called glucose. Glucose can then enter the cell through special molecules found in the membrane, called glucose transporters. Once inside the cell, glucose is broken down to make adenosine triphosphate (ATP), a form of energy, via two different pathways.

The first pathway, glycolysis, requires no oxygen and is referred to as anaerobic metabolism. Glycolysis occurs in the cytoplasm outside the mitochondria. During glycolysis, glucose is broken down into a molecule called pyruvate. Each reaction is designed to produce some hydrogen ions that can then be used to make energy packets (ATP). However, only four ATP molecules can be made from one molecule of glucose in this pathway. In prokaryotes, glycolysis is the only method used for converting energy.

the removal of a carbon and two oxygens, which go on to form carbon dioxide. When the carbon dioxide is removed, energy is given off, and a molecule called NAD+ is converted into the higher energy form, NADH. Another molecule, coenzyme A (CoA), then attaches to the remaining acetyl unit, forming acetyl CoA.

Acetyl CoA enters the Kreb's cycle by joining to a four-carbon molecule called oxaloacetate. Once the two molecules are joined, they make a six-carbon molecule called citric acid. Citric acid is then broken down and modified in a stepwise fashion. As this happens, hydrogen ions and carbon molecules are released. The carbon molecules are used to make more carbon dioxide. The hydrogen ions are picked up by NAD and another molecule called flavin-adenine dinucleotide (FAD). Eventually, the process produces the four-carbon oxaloacetate again, ending up where it started off. All in all, the Krebs’s cycle is capable of generating from 24 to 28 ATP molecules from one molecule of glucose converted to pyruvate. Therefore, it is easy to see how much more energy we can get from a molecule of glucose if our mitochondria are working properly and if we have oxygen.

Chloroplasts are similar to mitochondria but are found only in plants. A double membrane with an intermembrane space surrounds both organelles; both have their own DNA and are involved in energy metabolism; and both have reticulations, or many foldings, filling their inner spaces. Chloroplasts convert light energy from the sun into ATP through a process called photosynthesis.

Viruses are not classified as cells and therefore are neither unicellular nor multi-cellular organisms. Most people do not even classify viruses as "living" because they lack a metabolic system and are dependent on the host cells that they infect to reproduce. Viruses have genomes that consist of either DNA or RNA, and there are examples of viruses that are either double-stranded or single-stranded. Importantly, their genomes code not only for the proteins needed to package its genetic material but for those proteins needed by the virus to reproduce during its infective cycle.

For most unicellular organisms, reproduction is a simple matter of cell duplication, also known as replication. But for multi-cellular organisms, cell replication and reproduction are two separate processes. Multi-cellular organisms replace damaged or worn out cells through a replication process called mitosis, the division of a eukaryotic cell nucleus to produce two identical daughter nuclei. To reproduce, eukaryotes must first create special cells called gametes—eggs and sperm—that then fuse to form the beginning of a new organism. Gametes are but one of the many unique cell types that multi-cellular organisms need to function as a complete organism.

Most unicellular organisms create their next generation by replicating all of their parts and then splitting into two cells, a type of asexual reproduction called binary fission. This process spawns not just two new cells, but also two new organisms. Multi-cellular organisms replicate new cells in much the same way. For example, we produce new skin cells and liver cells by replicating the DNA found in that cell through mitosis. Yet, producing a whole new organism requires sexual reproduction, at least for most multi-cellular organisms. In the first step, specialized cells called gametes—eggs and sperm—are created through a process called meiosis. Meiosis serves to reduce the chromosome number for that particular organism by half. In the second step, the sperm and egg join to make a single cell, which restores the chromosome number. This joined cell then divides and differentiates into different cell types that eventually form an entire functioning organism.
 

These two cells then enter Meiosis II, producing four daughter nuclei, each with a single copy of each chromosome.

Meiosis is a specialized type of cell division that occurs during the formation of gametes. Although meiosis may seem much more complicated than mitosis, it is really just two cell divisions in sequence. Each of these sequences maintains strong similarities to mitosis.

Meiosis I refers to the first of the two divisions and is often called the reduction division. This is because it is here that the chromosome complement is reduced from diploid (two copies) to haploid (one copy). Interphase in meiosis is identical to interphase in mitosis. At this stage, there is no way to determine what type of division the cell will undergo when it divides. Meiotic division will only occur in cells associated with male or female sex organs. Prophase I is virtually identical to prophase in mitosis, involving the appearance of the chromosomes, the development of the spindle apparatus, and the breakdown of the nuclear membrane. Metaphase I is where the critical difference occurs between meiosis and mitosis. In mitosis, all of the chromosomes line up on the metaphase plate in no particular order. In Metaphase I, the chromosome pairs are aligned on either side of the metaphase plate. It is during this alignment that the chromatid arms may overlap and temporarily fuse, resulting in what is called crossovers. During Anaphase I, the spindle fibers contract, pulling the homologous pairs away from each other and toward each pole of the cell. In Telophase I, a cleavage furrow typically forms, followed by cytokinesis, the changes that occur in the cytoplasm of a cell during nuclear division; but the nuclear membrane is usually not reformed, and the chromosomes do not disappear. At the end of Telophase I, each daughter cell has a single set of chromosomes, half the total number in the original cell, that is, while the original cell was diploid; the daughter cells are now haploid.

Meiosis II is quite simply a mitotic division of each of the haploid cells produced in Meiosis I. There is no Interphase between Meiosis I and Meiosis II, and the latter begins with Prophase II. At this stage, a new set of spindle fibers forms and the chromosomes begin to move toward the equator of the cell. During Metaphase II, all of the chromosomes in the two cells align with the metaphase plate. In Anaphase II, the centromeres split, and the spindle fibers shorten, drawing

the chromosomes toward each pole of the cell. In Telophase II, a cleavage furrow develops, followed by cytokinesis and the formation of the nuclear membrane. The chromosomes begin to fade and are replaced by the granular chromatin, a characteristic of interphase. When Meiosis II is complete, there will be a total of four daughter cells, each with half the total number of chromosomes as the original cell. In the case of male structures, all four cells will eventually develop into sperm cells. In the case of the female life cycles in higher organisms, three of the cells will typically abort, leaving a single cell to develop into an egg cell, which is much larger than a sperm cell.

All organisms suffer a certain number of small mutations, or random changes in a DNA sequence, during the process of DNA replication. These are called spontaneous mutations and occur at a rate characteristic for that organism. Genetic recombination refers more to a large-scale rearrangement of a DNA molecule. This process involves pairing between complementary strands of two parental duplex, or double-stranded DNAs, and results from a physical exchange of chromosome material.

The position at which a gene is located on a chromosome is called a locus. In a given individual, one might find two different versions of this gene at a particular locus. These alternate gene forms are called alleles. During Meiosis I, when the chromosomes line up along the metaphase plate, the two strands of a chromosome pair may physically cross over one another. This may cause the strands to break apart at the crossover point and reconnect to the other chromosome, resulting in the exchange of part of the chromosome.

Recombination results in a new arrangement of maternal and paternal alleles on the same chromosome. Although the same genes appear in the same order, the alleles are different. This process explains why offspring from the same parents can look so different. In this way, it is theoretically possible to have any combination of parental alleles in an offspring, and the fact that two alleles appear together in one offspring does not have any influence on the statistical probability that another offspring will have the same combination. This theory of "independent assortment" of alleles is fundamental to genetic inheritance. However, having said that, there is an exception that requires further discussion.

The frequency of recombination is actually not the same for all gene combinations. This is because recombination is greatly influenced by the proximity of one gene to another. If two genes are located close together on a chromosome, the likelihood that a recombination event will separate these two genes is less than if they were farther apart. Linkage describes the tendency of genes to be inherited together as a result of their location on the same chromosome. Linkage disequilibrium describes a situation in which some combinations of genes or genetic markers occur more or less frequently in a population than would be expected from their distances apart. Scientists apply this concept when searching for a gene that may cause a particular disease. They do this by comparing the occurrence of a specific DNA sequence with the appearance of a disease. When they find a high correlation between the two, they know they are getting closer to finding the appropriate gene sequence.

Bacteria reproduce through a fairly simple process called binary fission, or the reproduction of a living cell by division into two equal, or near equal, parts. As just noted, this type of asexual reproduction theoretically results in two identical cells. However, bacterial DNA has a relatively high mutation rate. This rapid rate of genetic change is what makes bacteria capable of developing resistance to antibiotics and helps them exploit invasion into a wide range of environments.

Similar to more complex organisms, bacteria also have mechanisms for exchanging genetic material. Although not equivalent to sexual reproduction, the end result is that a bacterium contains a combination of traits from two different parental cells. Three different modes of exchange have thus far been identified in bacteria.

Conjunction involves the direct joining of two bacteria, which allows their circular DNA’s to undergo recombination. Bacteria can also undergo transformation by absorbing remnants of DNA from dead bacteria and integrating these fragments into their own DNA. Lastly, bacteria can exchange genetic material through a process called transduction, in which genes are transported into and out of the cell by bacterial viruses, called bacteriophages, or by plasmids, an autonomous self-replicating extra chromosomal circular DNA.

together by chance. This self-assembly is often aided by molecular chaperones, or proteins made by the host that help the capsid parts come together.There are three different ways genetic information contained in a viral genome can be reproduced. The form of genetic material contained in the viral capsid, the protein coat that surrounds the nucleic acid, determines the exact replication process. Some viruses have DNA, which once inside the host cell is replicated by the host along with its own DNA. Then, there are two different replication processes for viruses containing RNA. In the first process, the viral RNA is directly copied using an enzyme called RNA replicase. This enzyme then uses that RNA copy as a template to make hundreds of duplicates of the original RNA. A second group of RNA-containing viruses, called the retroviruses, uses the enzyme reverse transcriptase to synthesize a complementary strand of DNA so that the virus's genetic information is contained in a molecule of DNA rather than RNA. The viral DNA can then be further replicated using the host cell machinery.

Steps Associated with Viral Reproduction
1. Attachment, sometimes called absorption: The virus attaches to receptors on the host cell wall.
2. Penetration: The nucleic acid of the virus moves through the plasma membrane and into the cytoplasm of the host cell. The capsid of a phage, a bacterial virus, remains on the outside. In contrast, many viruses that infect animal cells enter the host cell intact.
3. Replication: The viral genome contains all the information necessary to produce new viruses. Once inside the host cell, the virus induces the host cell to synthesize the necessary components for its replication.
4. Assembly: The newly synthesized viral components are assembled into new viruses.
5. Release: Assembled viruses are released from the cell and can now infect other cells, and the process begins again

When the virus has taken over the cell, it immediately directs the host to begin manufacturing the proteins necessary for virus reproduction. The host produces three kinds of proteins: early proteins, enzymes used in nucleic acid replication; late proteins, proteins used to construct the virus coat; and lytic proteins, enzymes used to break open the cell for viral exit. The final viral product is assembled spontaneously, that is, the parts are made separately by the host and are joined together by chance. This self-assembly is often aided by molecular chaperones, or proteins made by the host that help the capsid parts come together.

The new viruses then leave the cell either by exocytosis or by lysis. Envelope-bound animal viruses instruct the host's endoplasmic reticulum to make certain proteins, called glycoproteins, which then collect in clumps along the cell membrane. The virus is then discharged from the cell at these exit sites, referred to as exocytosis. On the other hand, bacteriophages must break open, or lyse, the cell to exit. To do this, the phages have a gene that codes for an enzyme called lysozyme. This enzyme breaks down the cell wall, causing the cell to swell and burst. The new viruses are released into the environment, killing the host cell in the process.

Viruses are important to the study of molecular and cellular biology because they provide simple systems that can be used to manipulate and investigate the functions of many cell types. We have just discussed how viral replication depends on the metabolism of the infected cell. Therefore, the study of viruses can provide fundamental information about aspects of cell biology and metabolism. The rapid growth and small genome size of bacteria make them excellent tools for experiments in biology. Bacterial viruses have also further simplified the study of bacterial genetics and have deepened our understanding of the basic mechanisms of molecular genetics. Because of the complexity of an animal cell genome, viruses have been even more important in studies of animal cells than in studies of bacteria. Numerous studies have demonstrated the utility of animal viruses as probes for investigating different activities of eukaryotic cells. Other examples in which animal viruses have provided important models for biological research of their host cells include studies of DNA replication, transcription, RNA processing, and protein transport.

One family of animal viruses, called the retroviruses, contains RNA genomes in their virus particles but synthesize a DNA copy of their genome in infected cells. Retroviruses provide an excellent example of how viruses can play an important role as models for biological research. Studies of these viruses are what first demonstrated the synthesis of DNA from RNA templates, a fundamental mode for transferring genetic material that occurs in both eukaryotes and prokaryotes. other cells, and the process begins again.

Although much is known about transcript processing, the signals and events that instruct RNA polymerase to stop transcribing and drop off the DNA template remain unclear. Experiments over the years have indicated that processed eukaryotic messages contain a poly(A) addition signal (AAUAAA) at their 3' end, followed by a string of adenines. This poly(A) addition, also called the poly(A) site, contributes not only to the addition of the poly(A) tail but also to transcription termination and the release of RNA polymerase from the DNA template. Yet, transcription does not stop here. Rather, it continues for another 200 to 2000 bases beyond this site before it is aborted. It is either before or during this termination process that the nascent transcript is cleaved, or cut, at the poly(A) site, leading to the creation of two RNA molecules. The upstream portion of the newly formed, or nascent, RNA then undergoes further modifications, called post-transcriptional modification, and becomes mRNA. The downstream RNA becomes unstable and is rapidly degraded.

DNA Repair Mechanisms

Maintenance of the accuracy of the DNA genetic code is critical for both the long- and short-term survival of cells and species. Sometimes, normal cellular activities, such as duplicating DNA and making new gametes, introduce changes or mutations in our DNA. Other changes are caused by exposure of DNA to chemicals, radiation, or other adverse environmental conditions. No matter the source, genetic mutations have the potential for both positive and negative effects on an individual as well as its species. A positive change results in a slightly different version of a gene that might eventually prove beneficial in the face of a new disease or changing environmental conditions. Such beneficial changes are the cornerstone of evolution. Other mutations are considered deleterious, or result in damage to a cell or an individual. For example, errors within a particular DNA sequence may end up either preventing a vital protein from being made or encoding a defective protein. It is often these types of errors that lead to various disease states.

The potential for DNA damage is counteracted by a vigorous surveillance and repair system. Within this system, there are a number of enzymes capable of repairing damage to DNA. Some of these enzymes are specific for a particular type of damage, whereas others can handle a range of mutation types. These systems also differ in the degree to which they are able to restore the normal, or wild type, sequence.

Categories of DNA Repair Systems
· Photo-reactivation is the process whereby genetic damage caused by ultraviolet radiation is reversed by subsequent illumination with visible or near-ultraviolet light.
· Nucleotide excision repair is used to fix DNA lesions, such as single-stranded breaks or damaged bases, and occurs in stages. The first stage involves recognition of the damaged region. In the second stage, two enzymatic reactions serve to remove, or excise, the damaged sequence. The third stage involves synthesis by DNA polymerase of the excised nucleotides using the second intact strand of DNA as a template. Lastly, DNA lipase joins the newly synthesized segment to the existing ends of the originally damaged DNA strand.
Recombination repair, or post-replication repair, fixes DNA damage by a strand exchange from the other daughter chromosome. Because it involves homologous recombination, it is largely error free.
· Base excision repair allows for the identification and removal of wrong bases, typically attributable to de-amination—the removal of an amino group (NH2)—of normal bases as well as from chemical modification.
· Mismatch repair is a multi-enzyme system that recognizes inappropriately matched bases in DNA and replaces one of the two bases with one that "matches" the other. The major problem here is recognizing which of the mismatched bases is incorrect and therefore should be removed and replaced.
· Adaptive/inducible repair describes several protein activities that recognize very specific modified bases. They then transfer this modifying group from the DNA to themselves, and, in doing so, destroy their own function. These proteins are referred to as inducible because they tend to regulate their own synthesis. For example, exposure to modifying agents induces, or turns on, more synthesis and therefore adaptation.
· SOS repair or inducible error-prone repair is a repair process that occurs in bacteria and is induced, or switched on, in the presence of potentially lethal stresses, such as UV irradiation or the inactivation of genes essential for replication. Some responses to this type of stress include mutagenesis—the production of mutations—or cell elongation without cell division. In this type of repair process, replication of the DNA template is extremely inaccurate. Obviously, such a repair system must be a desperate recourse for the cell, allowing replication past a region where the wild-type sequence has been lost.
 

From Cells to Genomes

Understanding what makes up a cell and how that cell works is fundamental to all of the biological sciences. Appreciating the similarities and differences between cell types is particularly important to the fields of cell and molecular biology. These fundamental similarities and differences provide a unifying theme, allowing the principles learned from studying one cell type to be extrapolated and generalized to other cell types.

Perhaps the most fundamental property of all living things is their ability to reproduce. All cells arise from pre-existing cells, that is, their genetic material must be replicated and passed from parent cell to progeny. Likewise, all multi-cellular organisms inherit their genetic information specifying structure and function from their parents. The next section of the genetics primer, What is a Genome, http://www.ncbi.nlm.nih.gov/About/primer/genetics_genome.html details how genetic information is replicated and transmitted from cell to cell and organism-to-organism.

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