Biology Ch. 14 and 15 Test Topics

Gel Electrophoresis (Separating DNA)

Gel electrophoresis is used to separate DNA fragments. After being cut by restriction enzymes, the fragments are put into wells on a gel that is similar to a slice of gelatin. An electric voltage moves them across the gel. Shorter fragments move faster than longer fragments. Within an hour or two, the fragments all separate, each appearing as band on gel.

Restriction Endonucleases (Enzymes) (Cutting DNA)

However, DNA molecules from most organisms are much too large to be analyzed, so they must first be cut into smaller pieces. Many bacteria produce enzymes that do exactly that. Known as restriction enzymes, these highly specific substances cut even the largest DNA molecule into precise pieces, called restriction fragments, that are several hundred bases in length. Of the hundreds of known restriction enzymes, each cuts DNA at a different sequence of nucleotides. For example, A restriction enzyme is like a key that fits only one lock. The EcoRI restriction enzyme can only recognize the base sequence GAATTC. It cuts each strand of DNA between the G and A bases, leaving single-stranded overhangs with the sequence AATT. The overhangs are called "sticky ends" because they can bond, or "stick," to a DNA fragment with complementary base sequence.

Plasmids

In addition to their own large chromosomes, some bacteria contain small circular DNA molecules known as plasmids. Plasmids, are widely used in recombinant DNA studies. Joining DNA to a plasmid, and then using the recombinant plasmid to transform bacteria, results in the replication of the newly added DNA along with the rest of the cell's genome.

Reading DNA

After the DNA fragments have been separated, researchers use a clever chemical "trick" to read, or sequence, them. The single-stranded DNA fragments are placed in a test tube containing DNA polymerase—the enzyme that copies DNA—along with the four nucleotide bases, A, T, G, and C. As the enzyme goes to work, it uses the unknown strand as a template to make one new DNA strand after another. The tricky part is that researchers also add a small number of bases that have a chemical dye attached. Each time a dye-labeled base is added to a new DNA strand, the synthesis of that strand stops. When DNA synthesis is completed, the result is a series of color-coded DNA fragments of different lengths. Researchers can then separate these fragments, often by gel electrophoresis. The order of colored bands on the gel tells the exact sequence of bases in the DNA. The entire process can be automated and controlled by computers, so that DNA sequencing machines can read thousands of bases in a matter of seconds.

Selective Breeding

Allowing only those animals with wanted characteristics to produce the next generation. Humans use selective breeding, which takes advantage of naturally occurring genetic variation, to pass wanted traits on to the next generation of organisms.

Sex-Linked

Because the X and Y chromosomes determine sex, the genes located on them show a pattern of inheritance called sex-linkage. A sex-linked gene is a gene located on a sex chromosome. As you might expect, genes on the Y chromosome are found only in males and are passed directly from father to son. Genes located on the X chromosome are found in both sexes, but the fact that men have just one X chromosome leads to some interesting consequences.

Manipulating DNA (Beginnings)

Biologists dream of a time when they could read DNA sequences in human genome. DNA is a huge molecule—even the smallest human chromosome contains nearly 50 million base pairs. Manipulating such large molecules is extremely difficult. In the late 1960s, however, scientists found they could use natural enzymes in DNA analysis. From this discovery came many useful tools. By using tools that cut, separate, and then replicate DNA base by base, scientists can now read the base sequences in DNA from any cell. Such techniques have revolutionized genetic studies of living organisms, including humans.

DNA Microarrays

Even though all of the cells in the human body contain identical genetic material, the same genes are not active in every cell. By studying which genes are active and which are inactive in different cells, scientists can understand how the cells function normally and what happens when genes don't work as they should. Today, scientists use DNA microarray technology to study hundreds or even thousands of genes at once to understand their activity levels. A DNA microarray is a glass slide or silicon chip to which spots of single-stranded DNA have been tightly attached. Typically each spot contains a different DNA fragment. Different colored tags are used to label the source of DNA.

Human Genetic Disorders

Def: Thousands of genetic disorders caused by individual genes. These changes often affect specific proteins associated with important cellular functions. Sickle Cell Disease: This disorder is caused by a defective allele for beta-globin, one of two polypeptides in hemoglobin, the oxygen-carrying protein in red blood cells. The defective polypeptide makes hemoglobin a bit less soluble, causing hemoglobin molecules to stick together when the blood's oxygen level decreases. The molecules clump into long fibers, forcing cells into a distinctive sickle shape, which gives the disorder its name. Sickle-shaped cells are more rigid than normal red blood cells, and, therefore, they tend to get stuck in the capillaries—the narrowest blood vessels in the body. If the blood stops moving through the capillaries, damage to cells, tissues, and even organs can result. Cystic Fibrosis: Known as CF for short, cystic fibrosis is most common among people of European ancestry. CF is caused by a genetic change almost as small as the earwax allele. Most cases result from the deletion of just three bases in the gene for a protein called cystic fibrosis transmembrane conductance regulator (CFTR). CFTR normally allows chloride ions (Cl−) to pass across cell membranes. The loss of these bases removes a single amino acid—phenylalanine— from CFTR, causing the protein to fold improperly. The misfolded protein is then destroyed. With cell membranes unable to transport chloride ions, tissues throughout the body malfunction. People with one normal copy of the CF allele are unaffected by CF, because they can produce enough CFTR to allow their cells to work properly. Two copies of the defective allele are needed to produce the disorder, which means the CF allele is recessive. Children with CF have serious digestive problems and produce thick, heavy mucus that clogs their lungs and breathing passageways. Huntington's Disease: Huntington's disease is caused by a dominant allele for a protein found in brain cells. The allele for this disease contains a long string of bases in which the codon CAG—coding for the amino acid glutamine—repeats over and over again, more than 40 times. Despite intensive study, the reason why these long strings of glutamine cause disease is still not clear. The symptoms of Huntington's disease, namely mental deterioration and uncontrollable movements, usually do not appear until middle age. The greater the number of codon repeats, the earlier the disease appears, and the more severe are its symptoms. Chromosomal Disorders: Most of the time, the process of meiosis works perfectly and each human gamete gets exactly 23 chromosomes. Every now and then, however, something goes wrong. The most common error in meiosis occurs when homologous chromosomes fail to separate. This mistake is known as nondisjunction, which means "not coming apart." If nondisjunction occurs during meiosis, gametes with an abnormal number of chromosomes may result, leading to a disorder of chromosome numbers. For example, if two copies of an autosomal chromosome fail to separate during meiosis, an individual may be born with three copies of that chromosome. This condition is known as a trisomy, meaning "three bodies." The most common form of trisomy, involving three copies of chromosome 21, is Down syndrome, which is often characterized by mild to severe mental retardation and a high frequency of certain birth defects.

Types of Gene Transmission (Dominant and Recessive Alleles, Codominant and Multiple Alleles)

Dominant and Recessive Alleles: Many human traits follow a pattern of simple dominance. For instance, a gene known as MC1R helps determine skin and hair color. Some of MC1R's recessive alleles produce red hair. An individual with red hair usually has two of these recessive alleles, inheriting a copy from each parent. Dominant alleles for the MC1R gene help produce darker hair colors. Another trait that displays simple dominance is the Rhesus, or Rh blood group. The allele for Rh factor comes in two forms: Rh+ and Rh−. Rh+ is dominant, so an individual with both alleles (Rh+/ Rh−) is said to have Rh positive blood. Rh negative blood is found in individuals with two recessive alleles (Rh−/Rh−). Codominant and Multiple Alleles: The alleles for many human genes display codominant inheritance. One example is the ABO blood group, determined by a gene with three alleles: I A, I B, and i. Alleles I A and I B are codominant. They produce molecules known as antigens on the surface of red blood cells. As Figure 14-5 shows, individuals with alleles I A and I B produce both A and B antigens, making them blood type AB. The i allele is recessive. Individuals with alleles I AI A or I Ai produce only the A antigen, making them blood type A. Those with I BI B or I Bi alleles are type B. Those homozygous for the i allele (ii) produce no antigen and are said to have blood type O. If a patient has AB¬negative blood, it means the individual has I A and I B alleles from the ABO gene and two Rh− alleles from the Rh gene.

Extra Terms

Dominant: Dd. D IS DOMINANT, wins over d. Recessive: Tt. t IS RECESSIVE. Loses to T Multiple Alleles: So far, examples have described genes with two alleles such as a and A. Many genes exist in different forms and therefore said to have multiple alleles. Def: Gene with more than two alleles. Ex: Rabbit coat color determined by a single gene that has four different alleles. Homologous: Two sets of chromosomes, meaning each of four chromosomes from male parent have corresponding chromosome with female. Haploid is in full and Diploid is half of that.

Polymerase Chain Reaction

Once they find a gene, biologists often need to make many copies of it. A technique known as polymerase chain reaction (PCR) allows them to do exactly that. At one end of the original piece of DNA, a biologist adds a short piece of DNA that complements a portion of the sequence. At the other end, the biologist adds another short piece of complementary DNA. These short pieces are known as primers because they prepare, or prime, a place for DNA polymerase to start working. The first step in using the polymerase chain reaction method to copy a gene is to heat a piece of DNA, which separates its two strands. Then, as the DNA cools, primers bind to the single strands. Next, DNA polymerase starts copying the region between the primers. These copies can serve as templates to make still more copies. In this way, just a few dozen cycles of replication can produce billions of copies of the DNA between the primers.

Recombinant DNA

Recombinant DNA molecules are made up of DNA from different sources. Restriction enzymes cut DNA at specific sequences, producing "sticky ends," which are single stranded overhangs of DNA. If two DNA molecules are cut with the same restriction enzyme, their sticky ends will bond to a fragment of DNA that has the complementary sequence of bases. An enzyme known as DNA ligase can then be used to join the two fragments. The technology makes it possible to change genetic information.

Plasamid DNA Transformations

Scientists can insert a piece of DNA into a plasmid if both the plasmid and the target DNA have been cut by the same restriction enzymes to create sticky ends. With this method, bacteria can be used to produce human growth hormone. First, a human gene is inserted into bacterial DNA. Then, the new combination of genes is returned to a bacterial cell, which replicates the recombinant DNA over and over again.

DNA Fingerprinting

The complexity of the human genome ensures that no individual is exactly like any other genetically—except for identical twins, who share the same genome. Molecular biology has used this fact to develop a powerful tool called DNA fingerprinting for use in identifying individuals. DNA fingerprinting analyzes sections of DNA that may have little or no function but that vary widely from one individual to another. This method is shown in Figure 15-19. First, restriction enzymes cut a small sample of human DNA. Next, gel electrophoresis separates the restriction fragments by size. Then, a DNA probe detects the fragments that have highly variable regions, revealing a series of variously sized DNA bands. If enough combinations of enzymes and probes are used, the resulting pattern of bands can be distinguished statistically from that of any other individual in the world. DNA samples can be obtained from blood, sperm, or tissue—even from a hair strand if it has tissue at the root.

Autosomes

To distinguish them from the sex chromosomes, the remaining 44 human chromosomes are known as autosomal chromosomes, or autosomes. The complete human genome consists of 46 chromosomes, including 44 autosomes and 2 sex chromosomes. To quickly summarize the total number of chromosomes present in a human cell—both autosomes and sex chromosomes— biologists write 46,XX for females and 46,XY for males.

Karyotypes

To see human chromosomes clearly, cell biologists photograph cells in mitosis, when the chromosomes are fully condensed and easy to view. Scientists then cut out the chromosomes from the photographs and arrange them in a picture known as a karyotype. A karyotype shows the complete diploid set of chromosomes grouped together in pairs, arranged in order of decreasing size.

Sex Chromosomes

Two of the 46 chromosomes in the human genome are known as sex chromosomes, because they determine an individual's sex. Females have two copies of the X chromosome. Males have one X chromosome and one Y chromosome. This is the reason why males and females are born in a roughly 50 : 50 ratio. All human egg cells carry a single X chromosome (23,X). However, half of all sperm cells carry an X chromosome (23,X) and half carry a Y chromosome (23,Y). This ensures that just about half the zygotes will be males and half will be females. More than 1200 genes are found on the X chromosome, while Y chromosome is smaller and only carries 140 genes, most of which are associated with male sex determination and sperm development.