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Cell Nucleus: Within a eukaryotic cell, a membrane-limited body which contains chromosomes and one or more nucleoli (Cell nucleolus). The nuclear membrane consists of a double unit-type membrane which is perforated by a number of pores; the outermost membrane is continuous with the Endoplasmic reticulum. A cell may contain more than one nucleus. (From Singleton & Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d ed)

DNA Packaging

JoVE 10785

Eukaryotes have large genomes compared to prokaryotes. In order to fit their genomes into a cell, eukaryotes must pack their DNA tightly inside the nucleus. To do so, DNA is wound around proteins called histones to form nucleosomes, the main unit of DNA packaging. Nucleosomes then coil into compact fibers known as chromatin.

Most cells in the human body contain about 3 billion base pairs of DNA packaged into 23 pairs of chromosomes. It is hard to imagine exactly how much DNA these numbers represent. So how much packing has to happen to fit the genome into a cell? We can gain some insight by expressing the genome in terms of length. If we were to arrange the DNA of a single human cell, like a skin cell, into a straight line, it would be two meters long–over 6.5 feet. The human body contains around 50 trillion human cells. This means that each person has a total of about 100 trillion meters of DNA. In other words, each person has enough DNA to stretch from the Earth to the Sun 300 times! And humans do not have particularly large genomes–those of many fish, amphibians, and flowering plants are much larger. For example, the genome of the flowering plant Paris japonica is 25 times larger than the human diploid genome. These figures emphasize the astonishing task that eukaryotes must accomplish to pack their DNA inside cells.

 Core: Biology

Genomic DNA in Eukaryotes

JoVE 10760

Eukaryotes have large genomes compared to prokaryotes. To fit their genomes into a cell, eukaryotic DNA is packaged extraordinarily tightly inside the nucleus. To achieve this, DNA is tightly wound around proteins called histones, which are packaged into nucleosomes that are joined by linker DNA and coil into chromatin fibers. Additional fibrous proteins further compact the chromatin, which is recognizable as chromosomes during certain phases of cell division. Most cells in the human body contain about 6 billion base pairs of DNA packaged into 23 pairs of chromosomes. It is hard to imagine exactly how much DNA these numbers represent, and therefore it is difficult to grasp how densely packed DNA must be to fit into a cell. We can gain some insight by expressing the genome in terms of length. If we were to arrange the DNA of a single diploid cell into a straight line, it would be about two meters long! Note that humans do not have unusually large genomes. Many fish, amphibians, and flowering plants have much larger genomes than humans. For example, the haploid genome of the Japanese flowering plant Paris japonica contains about 50 times more DNA than the human haploid genome. These figures emphasize the astonishing work that histones and other chromatin remodeling proteins must do to package DNA.

 Core: Biology

Prokaryotic Cells

JoVE 10690

Prokaryotes are small unicellular organisms in the domains Archaea and Bacteria. Bacteria include many common organisms such as Salmonella and Escherichia coli, while the Archaea include extremophiles that live in harsh environments, such as volcanic springs.

Like eukaryotic cells, all prokaryotic cells are surrounded by a plasma membrane and have DNA that contains the genetic instructions, cytoplasm that fills the interior of the cell, and ribosomes that synthesize proteins. However, unlike eukaryotic cells, prokaryotes lack a nucleus or other membrane-bound intracellular organelles. Their cellular components generally float freely within the cytoplasm, although their DNA—usually consisting of a single, circular chromosome—is clustered within a region called the nucleoid. Inside the cytoplasm, many prokaryotes have small circular pieces of DNA called plasmids. These are distinct from the chromosomal DNA in the nucleoid and tend to have just a few genes—such as genes for antibiotic resistance. Plasmids are self-replicating and can be transmitted between prokaryotes. Most prokaryotes have a cell wall made of peptidoglycan that lies outside of their plasma membrane, which physically protects the cell and helps it maintain osmotic pressure in different environments. Many prokaryotes also have a sticky capsule layer that covers

 Core: Biology

Spermatogenesis

JoVE 10905

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of seminiferous tubules. These spermatogonial stem cells divide asymmetrically to give rise to additional stem cells (meaning that these structures “self-renew”), as well as sperm progenitors, called spermatocytes. Importantly, this method of asymmetric mitotic division maintains a population of spermatogonial stem cells in the male reproductive tract, ensuring that sperm will continue to be produced throughout a man’s lifespan. As spermatogenesis proceeds, spermatocytes embark on meiosis, and each ultimately divides to form four sperm—each with only 23 chromosomes— that are expelled into the male reproductive tract. Interestingly, this is in contrast to oogenesis in women, during which only a single egg is generated for every progenitor cell. At the end of spermatogenesis, sperm demonstrate their characteristic shape: a “head” harboring minimal cytoplasm and a highly condensed nucleus, as well as a motile tail (flagellum). They are small cells, with no organelles such as ribosomes, ER or Golgi, but do have many mitochondria around the flagellum for power. Just below the head is the acrosomal vesicle which contains hydrolytic enzymes to penetrate the egg outer coat—th

 Core: Biology

RNA Splicing

JoVE 10802

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

 Core: Biology

DNA Isolation

JoVE 10814

DNA from cells is required for many biotechnology and research applications, such as molecular cloning. To remove and purify DNA from cells, researchers use various methods of DNA extraction. While the specifics of different protocols may vary, some general concepts underlie the process of DNA extraction.

First, cells need to be lysed—broken open—to release the DNA into solution. Cells can be physically lysed using equipment such as a homogenizer, sonicator, or bead beater, which grind or otherwise apply force to break the cells open. Often, substances such as detergent are added during lysis to chemically disrupt the lipid-based cell membranes—helping release the DNA from the nucleus and cell. The spinning in a centrifuge sediments the cell debris to the bottom, and the lysate—containing cellular materials—is collected for further processing. The DNA must then be separated from other cellular molecules, such as RNA and proteins. Therefore, enzymes such as RNase and Proteinase K are often added during or after lysis to degrade RNA and proteins, respectively. Additionally, organic solvents such as phenol and chloroform are commonly used to separate DNA from protein. Typically, the sample is vortexed with phenol-chloroform and then centrifuged to separate the aqueous and organic phases in the tube. The DNA-containing aqueous p

 Core: Biology

Levels of Organization

JoVE 10648

Biological organization is the classification of biological structures, ranging from atoms at the bottom of the hierarchy to the Earth’s biosphere. Each level of the hierarchy represents an increase in complexity that builds upon the previous level.

The most basic levels include atoms, molecules, and biomolecules. Atoms, the smallest unit of ordinary matter, are composed of a nucleus and electrons. Molecules comprise two or more atoms held together by chemical bonds, most commonly covalent, ionic, or metallic bonds. Biomolecules are molecules found in living organisms, including proteins, nucleic acids, lipids, and carbohydrates. Biomolecules are often polymers—large molecules that are created from smaller, repeating units. For instance, proteins are composed of amino acids, and nucleic acids are composed of nucleotides. Biomolecules can be endogenous or exogenous. Endogenous means that the biomolecule is produced inside a living organism. Biomolecules can also be consumed; for example, a cow gets carbohydrates from digesting grass (exogenous), but the grass must produce the carbohydrates through photosynthesis (endogenous). The next hierarchical level comprises subcellular structures called organelles. Organelles are made up of biomolecules and compartmentalize eukaryotic cells. Organelle means “little organ” as

 Core: Biology

Gene Therapy

JoVE 10815

Gene therapy is a technique where a gene is inserted into a person’s cells to prevent or treat a serious disease. The added gene may be a healthy version of the gene that is mutated in the patient, or it could be a different gene that inactivates or compensates for the patient’s disease-causing gene. For example, in patients with severe combined immunodeficiency (SCID) due to a mutation in the gene for the enzyme adenosine deaminase, a functioning version of the gene can be inserted. The patient’s cells can then make the enzyme, curing this potentially deadly disease in some cases. Genes can be introduced into a patient’s cells in two main ways: in vivo—directly into a person through injection into specific tissues or into the bloodstream; and ex vivo—into cells that have been removed from the patient, which are transplanted back after the gene is inserted. The gene is usually inserted into a vector—often a virus that has been modified to not cause disease—to get the gene into the patient’s cells and delivered to the nucleus. In some cases—for instance, when retroviral vectors are used—the gene is randomly inserted into the person’s genome, leading to stable expression of the inserted gene. In others—such as when adenoviral vectors are used—the gene does not integrate i

 Core: Biology

X-Inactivation

JoVE 11002

The human X chromosome contains over ten times the number of genes as in the Y chromosome. Since males have only one X chromosome, and females have two, one might expect females to produce twice as many of the proteins, with undesirable results.

Instead, in order to avoid this potential issue, female mammalian cells inactivate nearly all the genes in one of their X chromosomes during early embryonic development. In the nuclear envelope surrounding the cell nucleus, the inactivated X chromosome condenses into a small, dense ball called a Barr body. In this state, most of the X-linked genes are not accessible to transcription. In placental mammals, the inactivated X chromosome—maternal or paternalā —is randomly determined (marsupials, however, preferentially inactivate the paternal X chromosome). X inactivation in one cell is also independent of X inactivation in other cells. Thus, about half the embryonic cells inactivate the maternal X copy; the remaining half inactivate the paternal copy, producing a mosaic. When these cells replicate, they produce cells with the same X chromosome inactivated. Notably, Barr bodies get reactivated in cells within the ovaries that become eggs. X inactivation accounts for the appearance of female tortoiseshell and calico cats. These cats are heterozygous for a gene with alleles for black fur and orange fur

 Core: Biology

Types of RNA

JoVE 10800

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

 Core: Biology
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