Resource Center (Aging) Part 3

Mitosis

Mitosis is divided into four stages: prophase, metaphase, anaphase, and telophase. The behavior and movement of the chromosomes characterize each stage. At prophase, DNA replication has already occurred and the nuclear membrane begins to break down. Condensation of the duplicated chromosomes initiates the phase (i.e. the very long, thin chromosomes are folded up to produce short, thick chromosomes that are easy to move and maneuver). Under the microscope the chromosomes become visible as X-shaped structures, which are the two duplicated chromosomes, often called sister chromatids. A special region of each chromosome, called a centromere, holds the chromatids together. Proteins bind to the centromere to form a structure called the kinetochore. The centrosome is duplicated, and the two migrate to opposite ends of the cell.

During metaphase the chromosomes are sorted out and aligned between the two centrosomes. By this time the nuclear membrane has completely broken down. The two centrosomes and the micro-tubules fanning out between them form the mitotic spindle. The area in between the spindles, where the chromosomes are aligned, is known as the metaphase plate. Some of the microtubules make contact with the kinetochores, while others overlap, with motor proteins situated in between.

Anaphase begins when the duplicated chromosomes move to opposite poles of the cell. The first step is the release of an enzyme that breaks the bonds holding the kinetochores together, thus allowing the sister chromatids to separate from each other while remaining bound to their respective microtubules. Motor proteins, using energy supplied by ATP, move along the microtubule dragging the chromosomes to opposite ends of the cell.


During telophase the daughter chromosomes arrive at the spindle poles and decondense to form the relaxed chromosomes characteristic of interphase nuclei. The nuclear envelope begins forming around the chromosomes, marking the end of mitosis. By the end of telophase individual chromosomes are no longer distinguishable and are referred to as chromatin. While the nuclear membrane reforms, a contractile ring, made of the proteins myosin and actin, begins pinching the parental cell in two. This stage, separate from mitosis, is called cytokinesis, and leads to the formation of two daughter cells, each with one nucleus.

Meiosis

Many eukaryotes reproduce sexually through the fusion of gametes (eggs and sperm). If gametes were produced mitotically, a catastrophic growth in the number of chromosomes would occur each time a sperm fertilized an egg. Meiosis is a special form of cell division that prevents this from happening by producing haploid gametes, each possessing half as many chromosomes as the diploid cell. When haploid gametes fuse, they produce an embryo with the correct number of chromosomes.

Unlike mitosis, which produces two identical daughter cells, meiosis produces four genetically unique daughter cells that have half the number of chromosomes found in the parent cell. This is possible because meiosis consists of two rounds of cell division, called meiosis I and meiosis II, with only one round of DNA synthesis. Microbiologists discovered meiosis almost 100 years ago by comparing the number of chromosomes in somatic cells and germ cells. The roundworm, for example, was found to have four chromosomes in its somatic cells, but only two in its gametes. Many other studies also compared the amount of DNA in nuclei from somatic cells and gonads, always with the same result: The amount of DNA in somatic cells is at least double the amount in fully mature gametes.

Meiotic divisions are divided into the four mitotic stages discussed above. Indeed, meiosis II is virtually identical to a mitotic division. Meiosis I resembles mitosis, but close examination shows two important differences: Gene swapping occurs between homologous chromosomes in prophase, producing recombinant chromosomes, and the distribution of maternal and paternal chromosomes to different daughter cells. At the end of meiosis I, one of the daughter cells contains a mixture of normal and recombinant maternal chromosomes, and the other contains normal and recombinant paternal chromosomes. During meiosis II, the duplicated chromosomes are distributed to different daughter cells, yielding four, genetically unique cells: paternal, paternal recombinant, maternal, and maternal recombinant. Mixing genetic material in this way is unique to meiosis, and it is one of the reasons sexual reproduction has been such a powerful evolutionary force.

Cell Communication

A forest of glycoproteins and glycolipids covers the surface of every cell like trees on the surface of the Earth. The cell’s forest is called the glycocalyx, and many of its trees function like sensory antennae. Cells use these antennae to communicate with their environment and with other cells. In multicellular organisms the glycocalyx also plays an important role in holding cells together. In this case the antennae of adjacent cells are connected to one another through the formation of chemical bonds.

The sensory antennae, also known as receptors, are linked to a variety of secondary molecules that serve to relay messages to the interior of the cell. These molecules, some of which are called second messengers, may activate machinery in the cytoplasm, or they may enter the nucleus to activate gene expression. The signals that a cell receives are of many different kinds, but generally fall into one of five categories: 1) proliferation, which stimulates the cell to grow and divide; 2) activation, which is a request for the cell to synthesize and release specific molecules; 3) deactivation, which serves as a brake for a previous activation signal; 4) navigation, which helps direct the cell to a specific location (this is very important for free-living cells hunting for food and for immune system cells that are hunting for invading microorganisms); 5) termination, which is a signal that orders the cell to commit suicide. This death signal occurs during embryonic development (e.g. the loss of webbing between the fingers and toes) and during an infection. In some cases the only way the immune system can deal with an invading pathogenic microbe is to order some of the infected cells to commit suicide. This process is known as apoptosis.

Biotechnology

Biotechnology (also known as recombinant DNA technology) consists of several procedures that are used to study the structure and function of genes and their products. Central to this technology is the ability to clone specific pieces of DNA and to construct libraries of these DNA fragments that represent the genetic repertoire of an entire organism or a specific cell type. With these libraries at hand, scientists have been able to study the cell and whole organisms in unprecedented detail. The information so gained has revolutionized biology as well as many other disciplines, including medical science, pharmacology, psychiatry, and anthropology, to name but a few.

DNA cloning

In 1973 scientists discovered that restriction enzymes (enzymes that can cut DNA at specific sites), DNA ligase (an enzyme that can join two pieces of DNA together), and bacterial plasmids could be used to clone DNA molecules. Plasmids are small (about 3,000 base pairs) circular minichromosomes that occur naturally in bacteria and are often exchanged between cells by passive diffusion. A bacterium is said to be transfected when it acquires a new plasmid. For bacteria, the main advantage to swapping plasmids is that they often carry antibiotic resistance genes, so that a cell sensitive to ampicillin can become resistant simply by acquiring the right plasmid. For scientists, plasmid swapping provided an ideal method for amplifying or cloning a specific piece of DNA.

The first cloning experiment used a plasmid from the bacterium Escherichia coli that was cut with the restriction enzyme EcoRI. The plasmid had a single EcoRI site, so the restriction enzyme simply opened the circular molecule. Foreign DNA, cut with the same restriction enzyme, was incubated with the plasmid. Because the plasmid and foreign DNA were both cut with EcoRI, the DNA could insert itself into the plasmid to form a hybrid, or recombinant plas-mid, after which DNA ligase sealed the two together. The reaction mixture was added to a small volume of E. coli so that some of the cells could take up the recombinant plasmid before being transferred to a nutrient broth containing streptomycin.

Biotechnology. This technology consists of six basic steps: 1) digestion of DNA with restriction enzymes in order to isolate specific DNA fragments; 2) cloning of restriction fragments in circular bacterial minichro-mosomes to increase their numbers; 3) storing the fragments for further study in viral-based DNA libraries; 4) isolation and purification of DNA fragments from gene libraries using gel electrophoresis; 5) sequencing cloned DNA fragments; 6) determining the expression profile of selected DNA clones using RNA blots and radioactive detection procedures.

Biotechnology. This technology consists of six basic steps: 1) digestion of DNA with restriction enzymes in order to isolate specific DNA fragments; 2) cloning of restriction fragments in circular bacterial minichro-mosomes to increase their numbers; 3) storing the fragments for further study in viral-based DNA libraries; 4) isolation and purification of DNA fragments from gene libraries using gel electrophoresis; 5) sequencing cloned DNA fragments; 6) determining the expression profile of selected DNA clones using RNA blots and radioactive detection procedures.

Only those cells carrying the recombinant plasmid, which contained an antistrepto-mycin gene, could grow in the presence of this antibiotic. Each time the cells divided, the plasmid DNA was duplicated along with the main chromosome. After the cells had grown overnight, the foreign DNA had been amplified billions of times and was easily isolated for sequencing or expression studies. In this procedure the plasmid is known as a cloning vector because it serves to transfer the foreign DNA into a cell.

DNA Libraries

The basic cloning procedure described above not only provides a way to amplify a specific piece of DNA, but it can also be used to construct DNA libraries. In this case, however, the cloning vector is a bacteriophage called lambda. The lambda genome is double-stranded DNA of about 40,000 base pairs (bp), much of which can be replaced by foreign DNA without sacrificing the ability of the virus to infect bacteria. This is the great advantage of lambda over a plasmid. Lambda can accommodate very long pieces of DNA, often long enough to contain an entire gene, whereas a plasmid cannot accommodate foreign DNA that is larger than 2,000 base pairs. Moreover, a bacteriophage has the natural ability to infect bacteria, so that the efficiency of transfection is 100 times greater than it is for plasmids.

The construction of a DNA library begins with the isolation of genomic DNA and its digestion with a restriction enzyme to produce fragments of 1,000 to 10,000 bp. These fragments are ligated into lambda genomes, which are subjected to a packaging reaction to produce mature viral particles, most of which carry a different piece of the genomic DNA. This collection of viruses is called a ge-nomic library and is used to study the structure and organization of specific genes. Clones from a library such as this contain the coding sequences, in addition to noncoding sequences such as introns, intervening sequences, promoters, and enhancers. An alternative form of a DNA library can be constructed by isolating messenger RNA (mRNA) from a specific cell type. This RNA is converted to the complementary DNA (cDNA) using an RNA-dependent DNA polymerase called reverse transcriptase. The cDNA is ligated to lambda genomes and packaged as for the genomic library. This collection of recombinant viruses is known as a cDNA library and contains genes that were being expressed by the cells when the mRNA was extracted. It does not include introns or controlling elements as these are lost during transcription and the processing that occurs in the cell to make mature mRNA. Thus a cDNA library is intended for the purpose of studying gene expression and the structure of the coding region only.

Labeling cloned DNA

Many of the procedures used in biotechnology were inspired by the events that occur during DNA replication (described above). This includes the labeling of cloned DNA for use as probes in expression studies, DNA sequencing, and PCR (described below). DNA replication involves duplicating one of the strands (the parent, or template strand) by linking nucleotides in an order specified by the template and depends on a large number of enzymes, the most important of which is DNA polymerase. This enzyme, guided by the template strand, constructs a daughter strand by linking nucleo-tides together. One such nucleotide is deoxyadenine triphosphate (dATP). Deoxyribonucleotides have a single hydroxyl group located at the 3′ carbon of the sugar group while the triphosphate is attached to the 5′ carbon.

The procedure for labeling DNA probes, developed in 1983, introduces radioactive nucleotides into a DNA molecule. This method supplies DNA polymerase with a single-stranded DNA template, a primer, and the four nucleotides in a buffered solution to induce in vitro replication. The daughter strand, which becomes the labeled probe, is made radioactive by including a 32P-labeled nucleotide in the reaction mix. The radioactive nucleotide is usually deoxy-cytosine triphosphate (dCTP) or dATP. The 32P is always part of the a (alpha) phosphate (the phosphate closest to the 5′ carbon), as this is the one used by the polymerase to form the phosphodiester bond between nucleotides. Nucleotides can also be labeled with a fluorescent dye molecule.

Single-stranded DNA hexamers (six bases long) are used as primers, and these are produced in such a way that they contain all possible permutations of four bases taken six at a time. Randomizing the base sequence for the primers ensures that there will be at least one primer site in a template that is only 50 bp long. Templates used in labeling reactions such as this are generally 100 to 800 bp long. This strategy of labeling DNA is known as random primer labeling.

Gel Electrophoresis

This procedure is used to separate DNA and RNA fragments by size in a slab of agarose (highly refined agar) or polyacrylamide subjected to an electric field. Nucleic acids carry a negative charge and thus will migrate toward a positively charged electrode. The gel acts as a sieving medium that impedes the movement of the molecules. Thus the rate at which the fragments migrate is a function of their size; small fragments migrate more rapidly than large fragments. The gel containing the samples is run submerged in a special pH-regulated solution.Agarose gels are run horizontal as shown in the figure. But DNA sequencing gels, made of polyacryl-amide, are much bigger and are run in a vertical tank.

DNA Sequencing

A sequencing reaction developed by the British biochemist Dr. Fred Sanger in 1976, is a technique that takes its inspiration from the natural process of DNA replication. DNA polymerase requires a primer with a free 3′ hydroxyl group. The polymerase adds the first nucleotide to this group, and all subsequent bases are added to the 3′ hydroxyl of the previous base. Sequencing by the Sanger method is usually performed with the DNA cloned into a special sequencing plasmid. This simplifies the choice of the primers since their sequence can be derived from the known plasmid sequence. Once the primer binds to the primer site, the cloned DNA may be replicated.

Sanger’s innovation involved the synthesis of chain-terminating nucleotide analogues lacking the 3′ hydroxyl group. These analogues, also known as dideoxynucleotides (ddATP, ddCTP, ddGTP and ddTTP), terminate the growth of the daughter strand at the point of insertion, and this can be used to determine the distance of each base on the daughter strand from the primer. These distances can be visualized by separating the Sanger reaction products on a polyacrylamide gel, and then exposing the gel to X-ray film to produce an autoradiogram. The DNA sequence is read directly from this film, beginning with the smallest fragment at the bottom of the gel (the nucleotide closest to the primer), and ending with the largest fragment at the top.The smallest fragment in this example is the "C" nucleotide at the bottom of lane 3. The next nucleotide in the sequence is the "G" nucleotide in lane 4, then the "T" nucleotide in lane 2, and so on to the top of the gel.

Automated versions of the Sanger sequencing reaction use fluorescent-labeled dideoxynucleotides, each with a different color, so a computer can record the sequence of the template as the reaction mix passes a sensitive photocell. Machines such as this were used to sequence the human genome, a job that cost many millions of dollars and took years to complete. Recent advances in DNA sequencing technology will make it possible to sequence the human genome in less than a week at a cost of $1,000.

Gene Expression

The production of a genomic or cDNA library, followed by the sequencing of isolated clones, is a very powerful method for characterizing genes and the genomes from which they came. But the icing on the cake is the ability to determine the expression profile for a gene: That is, to determine which cells express the gene and exactly when the gene is turned on and off. Typical experiments may wish to determine the expression of specific genes in normal versus cancerous tissue, or tissues obtained from groups of different ages. There are essentially three methods for doing this: RNA blotting, Fluorescent In Situ Hybridization (FISH), and the Polymerase Chain Reaction.

RNA Blotting

This procedure consists of the following steps:

1. Extract mRNA from the cells or tissue of interest.

2. Fractionate (separate by size) the mRNA sample using gel electrophoresis.

3. Transfer the fractionated sample to a nylon membrane (the blotting step).

4. Incubate the membrane with a gene fragment (usually a cDNA clone) that has been labeled with a radioisotope.

5. Expose the membrane to X-ray film to visualize the signal.

The RNA is transferred from the gel to a nylon membrane using a vacuum apparatus or a simple dish containing a transfer buffer topped by a large stack of ordinary paper towels and a weight. The paper towels pull the transfer buffer through the gel, eluting the RNA from the gel and trapping it on the membrane. The location of specific mRNAs can be determined by hybridizing the membrane to a radiolabeled cDNA or genomic clone. The hybridization procedure involves placing the membrane in a buffer solution containing a labeled probe. During a long incubation period, the probe binds to the target sequence immobilized on the membrane. A-T and G-C base pairing (also known as hybridization) mediate the binding between the probe and target. The double-stranded molecule that is formed is a hybrid, being formed between the RNA target, on the membrane, and the DNA probe.

Fluorescent In Situ Hybridization (FISH)

Studying gene expression does not always depend on RNA blots and membrane hybridization. In the 1980s scientists found that cDNA probes could be hybridized to DNA or RNA in situ, that is, while located within cells or tissue sections fixed on a microscope slide. In this case the probe is labeled with a fluorescent dye molecule, rather than a radioactive isotope. The samples are then examined and photographed under a fluorescent microscope. FISH is an extremely powerful variation on RNA blotting. This procedure gives precise information regarding the identity of a cell that expresses a specific gene, information that usually cannot be obtained with membrane hybridization. Organs and tissues are generally composed of many different kinds of cells, which cannot be separated from one another using standard biochemical extraction procedures. Histologi-cal sections, however, show clearly the various cell types, and when subjected to FISH analysis, provide clear information as to which cells express specific genes. FISH is also used in clinical laboratories for the diagnosis of genetic abnormalities.

Next post:

Previous post: