Computer generated monophyletic clades based on chloroplast DNA have resulted in drastic changes to the Scrophulariaceae. Plantago, Penstemon, Veronica, Linaria, Antirrhinum, Keckiella, & Digitalis are now placed in the Plantaginaceae. Mimulus with its thigmotrophic stigma is placed in the Phrymaceae. Traditional genera retained in the Scropulariaceae include Verbascum and Scrophularia. Other genera placed in the Scrophulariaceae include Buddleja and Myoporum. Indian paintbrush (Castilleja), Indian warrior (Pedicularis), and owl's clover (Orthocarpus) are placed in the parasitic family Orobanchaceae with the broomrapes (Orobanche). Other closely-related families representing separate clades are the Paulowniaceae, Lentibulariaceae, Acanthaceae and Bignoniaceae. Members of the Martyniaceae (Proboscidea, Martynia & Ibicella) are included in the Pedaliaceae with Uncarina, Harpagophytum & Sesamum.

The Human Genome Project is a worldwide endeavor to map the DNA base sequence of every gene in the human genome. As of February 2001, the total number of functional genes is considerably less than expected, about 30,000 genes per cell compared with previous estimates of 100,000 genes. It has been estimated that a human somatic cell contains about 5 billion base pairs. If the average gene contains 1500 bases, then 30,000 functional genes is only about one percent of the total DNA per cell. Although there is an estimated six feet of DNA per human cell, only a small fraction of this amount consititutes the actual protein-coding genes.

Much of the DNA of humans is referred to as "variable number tandem repeats" (VNTRs) rather than specific protein-coding genes. The greatest variation in the DNA of two individuals is not in the protein-coding genes, but in the nonprotein-coding sections of their DNA. Natural selection has resulted in some time-tested DNA sequences called genes which are identical in normal individuals. The exact number and order of amino acids in protein molecules are determined by the DNA base sequences of genes, and genetic mutations are essentially "misspelled " genes. Genetic mutations, including variations in the base sequences of vital genes, may be fatal if they fail to code for a vital enzyme. For example, the dominant gene for hemoglobin is a time-tested sequence of DNA bases that is essential for the production of this life-giving pigment. Hemoglobin is a quaternary protein composed of four polypeptides and 484 amino acids. The substitution of valine for glutamic acid (glutamate) in the beta polypeptide changes the oxygen-carrying potential of this vital blood cell pigment, and is the biochemical explanation for the genetic disease called sickle-cell anemia. Natural selection does not limit variability in nonprotein-coding sections since these regions of DNA are not involved in the survival or reproductive success of individuals. Consequently, the DNA used to show variation between individuals comes from the nonprotein-coding sections called VNTRs. DNA sections unique to each individual are separated in a process called gel electrophoresis using a gel box.

DNA is negatively charged and migrates to the positive pole of a gel box containing agarose gel. The porous gel is made from agar, a polysaccharide extract from red algae (division Rhodophyta). Precise amounts of the DNA solutions being compared (containing RFLPs) are transferred to indentations or wells in the gel using a micropipetter. Gel patterns are similar to chromatographs and the process of separating sections of DNA is called gel electrophoresis. Restriction enzymes cut DNA into sections or fragments called restriction fragment length polymorphisms (RFLPs). Restriction enzymes are analogous to molecular scissors, cutting the DNA at specific base sequences called restriction sites. These enzymes were originally discovered in bacteria, a remarkable defensive mechanism that enables bacteria to cleave invading viral DNA, thus rendering it harmless. One restriction enzyme can cut DNA into more than 700,000 pieces. For example, a specific restriction enzyme (Hind3) cuts the DNA between adenine and adenine on the base sequence A|AGCTT. One DNA strand runs in the 5' to 3' direction, while the complementary strand runs in the 3' to 5' direction. The complementery strand is also cut between adenine and adenine TTCGA|A. Another restriction enzyme (EcoR2) cuts the DNA between guanine and adenine on the base sequence G|AATTC. The complementary strand is also cut between adenine and guanine CTTAA|G. The exact base sequence and length of a DNA fragment varies with different individuals. Every person has fragments with different lengths and unique base patterns, such as AGCTT and AATTC. The following table summarizes how the restriction enzymes Hind3 and EcoR2 cut specific base sequences at specific retriction sites.

Because of different numbers of purine and pyrimidine bases, the DNA fragments (RFLPs) have different molecular weights and migrate to different positions in the gel box. The fragments are displayed as bands in the gel, similar to the separation of different molecules in chromatography. Gel electrophoresis can separate DNA molecules that differ in length by only a few nucleotides. Banding patterns can be enhanced when viewed on a light box or under ultraviolet light. They may also be photographed. The specific banding pattern of an individual depends on the precise fragments that are separated on a gel layer. Because everyone has slightly different banding patterns, gel electrophoresis is used to determine the precise DNA fingerprint of an individual. In a human DNA fingerprint, thousands of bands from the evidence (crime scene) and suspect are carefully compared in order to show a percent similarity. DNA fragments (RFLPs) can also be anayzed from plants, algae and fungi. In order to run a sufficient quantity of fragments to produce a visible banding pattern, the DNA is amplified using the PCR technique (polymerase chain reaction) described below.

Gender verification in the Olympic Games now employs sophisticated DNA testing rather than counting Barr bodies within the nuclei of cells. The test is designed to detect the presence of the SRY gene (sex region Y chromosome), a region of DNA on the short arm of the Y chromosome responsible for masculinization of the fetus. Cells from the buccal mucosa (squamous epithelial cells), often called "cheek cells" in general biology classes, are obtained by gently scraping the inside of the mouth with a toothpick. The DNA in the nuclei of these cells is amplified using the PCR technique (polymerase chain reaction). If present, the SRY gene will show up as a unique banding pattern by electrophoresis on agar gels.

In addition to DNA fingerprinting based on banding patterns from gel electrophoresis, scientists can also determine the exact sequence of bases (adenine, thymine, guanine and cytosine) in a DNA fragment or a complete gene. An instrument called an automated DNA sequencer analyzes the DNA sample and produces a printout with peaks and valleys representing all the four nucleotides (A, T, C and G). Special fluorescent nucleotides amplified with PCR produce color-coded printouts of the four bases. Modified nucleotides used in the PCR replication contain an attached molecule that fluoresces a particular color when it passes through a laser beam. Each DNA fragment (band) extracted through gel electrophoresis can be sequenced to show the exact order of bases. Entire genes are also sequenced, including DNA from chloroplasts, mitochondria, introns, and the genes that translate for large and small subunits of ribosomes.

DNA sequencing is a valuable tool in taxonomic studies of species within families and the phylogenetic relationships of larger categories of animals and plants. One of the most interesting studies of human genes has resulted in a theory that traces the mitochondrial DNA of humans to an ancestral woman who lived in Africa about 100,000 years ago. Since mitochondria are only passed on through the egg, the genes are relatively stable from generation to generation, compared with nuclear DNA which may be altered during meiosis and sexual reproduction. Chromosomal genes are recombined during crossing over, and reshuffled during random assortment of the chromosomes and random combination of the gametes.

All all of these remarkable methods of DNA analysis would be impossible without the ability to amplify DNA with the polymerase chain reaction. PCR is an extremely valuable technique in forensic criminology involving rape, murder and disputed parentage. DNA can be identified from small samples of blood, saliva, skin, hair follicles and semen. In fact, the acronym PCR became well-known during the O.J. Simpson trial. When amplifying genes using PCR, it is imperative that the sample not be contaminated with any foreign DNA, otherwise the foreign genes may be inadvertently amplified. For example, in a research paper the genes of a spruce tree were sequenced, only to find out later that the actual DNA came from an internal parasitic fungus that was living within the spruce sample!

In order to amplify a gene using PCR, the DNA from an organism must be extracted and placed in a test tube. There are several "cookbook method" procedures for extracting nuclear DNA from the nucleus and nucleolus, and cytoplasmic DNA from cellular organelles, including the chloroplast and mitochondria. Total genomic DNA includes the nucleus and cytoplasmic organelles. The following procedure was used to isolate genomic DNA from a duckweed (Lemna minuta). Although members of the duckweed family (Lemnaceae) are commonly polyploid, they still have one genome composed of multiple sets of chromosomes.

DNA can easily be extracted from dried split peas (or other vegetables) and a few ordinary household chemicals, including liquid detergent, meat tenderizer and rubbing alcohol (isopropyl alcohol). The procedure is illustrated at the web site of the Genetic Science Learning Center, University Of Utah:

A thick, pea-cell soup is made by grinding up 100 ml of dried split peas in a blender with 200 ml water, and then filtering through a fine-mesh strainer into a measuring cup. Two tablespoons of liquid detergent are added and the soup is allowed to sit for 10 minutes. Next the soup is placed in test tubes (1/3 full) or small glass containers. There is sufficient pea soup to fill a dozen or more small test tubes up to 1/3 full. Then a pinch of meat tenderizer containing papain or bromelain enzymes is added to each test tube and the mixture is gently (briefly) stirred with a slender rod such as a wooden skewer. Finally, rubbing alcohol (70-90% isopropyl alcohol) is slowly pored into each test tube (2/3 full) so that it forms a layer on top of the pea mixture. DNA rises into the alcohol layer like a cottony mass of threads and can be rolled onto a wooden stick or stirring rod. By adding isopropanol (which is infinitely soluble in the aqueous layer but not the DNA), the resultant solution forces the DNA out of solution as a solid. I.e. when the concentration of alcohol/water solution is such that the DNA is no longer soluble, the DNA precipitates out of solution. If you don't see any DNA, let the test tube sit for 15 minutes to an hour. A cottony mass should be visible in the alcohol layer, just above the thick pea soup layer. This genomic DNA comes from all the cells of the ground up peas. Unless it is cut by restriction enzymes into sections (RFLPs), it is much too long and stringy to migrate through the pores of agarose gel during electrophoresis.

An interesting fact about enzymes is that JelloŽ is not recommended with the following fresh or frozen fruits and roots: pineapple (Bromeliaceae: Ananas comosus), papaya (Caricaceae: Carica papaya), figs (Moraceae: Ficus carica), guava (Myrtaceae: Psidium guajava), kiwi (Actinidiaceae: Actinidia chinensis), and ginger root (Zingiberaceae: Zingiber officinale). All of these plants contain proteolytic (protein digesting) enzymes which prevent the gelatin from setting (changing into a gel state) as it cools. Some of these protease enzymes have been used medicinally and as meat tenderizers, such as ficin from figs (Ficus), papain from papaya (Carica), and bromelain from pineapples (Ananas). Try adding some pineapple juice to milk. The milk protein begins to coagulate and degrade as it reacts with the bromelain. Pineapple juice will also remove the gelatin-emulsion surface on black & white photographic film. [The emulsion surface contains light sensitive silver halides in a gelatin that is rinsed away during processing. The silver that remains on the film emulsion reveals the negative image from which the photographic print is created.] In French Polynesia, the ficin-rich sap from a native banyan fig is used to kill parasitic worms and to treat worts and skin cancers. Ficin also breaks down the female pollinator wasp inside wasp-pollinated Calimyrna figs grown in California's Central Valley. When you eat one of these delicious figs, you won't find the wasp inside that was responsible for the seed formation and superior nutty flavor.

DNA polymerase and a mixture of all four nucleotides are added to a test tube containing the extracted DNA sample. When the double-stranded parental (template) DNA is heated to 95 degrees Celsius, the individual strands unwind and separate from each other. The objective is to replicate the section of each strand containing the target gene using the enzyme DNA polymerase. Each single parental strand of DNA has the remarkable property of rebuilding the missing complementary strand as nucleotides attach in the 5 prime (5') to 3 prime (3') direction. Each newly-formed complementary strand (one for each parental strand) is called a "daughter strand."

In order for DNA polymerase to find the start of a specific target gene in each section of DNA, a short segment of DNA called a primer must be attached (annealed) to each "mother" DNA strand upstream (toward 3' end) from each gene. The primer does not overlap the target gene, because it is complementary to the base sequence that appears just before the gene on the mother strand of DNA. The complementary "daughter" strand is produced in the 5' to 3' direction. Primers contain about 20 bases and they have been synthesized for many of the genes that are commonly amplified using the PCR technique. They may be purchased from biotechnology supply companies. The primer for a specific gene is added to the mixture of single-stranded DNA after it has cooled down to 52-54^o C (126-129^o F).

As the double-stranded, parental (template) DNA ladder unzips and nucleotides attach to each of the two single parental strands, something very interesting happens. One daughter strand, called the "leading strand," forms continuously as nucleotides attach in the 5' to 3' direction. But in the other daughter strand, called the "lagging strand," the nucleotides attach in discontinuous sections. These sections are called Okazaki fragments, named after the Japanese scientist Reiji Okazaki who discovered them. Since the lagging strand is complementary to the leading strand, its 3' end is opposite the leading strand's 5' end, and vice versa. The only way this strand can lengthen in the 5' to 3' direction as the parental DNA molecule unzips, is is for it to grow in sections or fragments. This remarkable discovery is shown in the following illustration.

The DNA mixture is heated to 72^o C (162^o F) and DNA polymerase recognizes the primer annealed to each strand and proceeds to synthesize the complementary strand all the way down the gene. Nucleotides attach along the gene from all the adenines, thymines, cytosines and guanines that are already in the mixture. Now the mixture contains two identical copies of the gene (two complete DNA ladders). DNA polymerase from the bacterium Thermus aquaticus (called TAQ polymerase) is used for the reaction because it is immune to the high temperatures. Unlike most protein enzymes that are destroyed at temperatures above 40^o C (104^o F), DNA polymerase from Thermus aquaticus can survive the 72^o C of the reaction. In fact, this bacterium normally lives in hot springs and can survive temperatures approaching the boiling point of water.

Boiling hot springs in Yellowstone National Park are colored by colonies of thermophilic cyanobacteria, eubacteria and archaebacteria. Orange-colored cyanobacteria generally occur in water that has cooled below 73^o C (163^o F). The green chlorophylls in these photosynthetic bacteria are masked by orange carotenoid pigments. Like the bright red halobacteria of salt lakes, carotenoids protect the delicate cells from intense solar radiation, especially during the summer months. Warmer, whitish areas of the ponds contain stringy masses of nonphotosynthetic eubacteria. Thermus aquaticus survives in temperatures too high for photosynthetic bacteria, up to 80^o C (176^o F). Thermus aquaticus is heterotrophic and survives on minute amounts of organic matter in the water. TAQ polymerase used in the amplification of DNA using the polymerase chain reaction (PCR) was originally isolated from a colony of T. aquaticus collected in a hot spring at Yellowstone National Park.

Archaebacteria thrive in boiling water at Yellowstone National Park, at temperatures of 92^o C (198^o F). These bacteria also thrive near steam vents at the bottom of the ocean at temperatures exceeding 115^o C (239^o F). Scientists from throughout the world are studying the amazing bacteria flora at Yellowstone National Park. This is one of the best places on earth to study these organisms in their natural protected habitats. In other parts of the world, similar hot springs have been destoyed for the production of geothermal energy.

Acid hot springs in Yellowstone National Park with a pH of below 4.0 support the eukaryotic alga Cyanidium caldarum. This remarkable photosynthetic alga can even survive in a pH of zero! Some acidophilic hot springs bacteria utilize the oxidation of sulfur and iron for the synthesis of ATP. Alkaline hot springs support colonies of bacteria that utilize hydrogen sulfide for their energy source.

Life as we know it may have first arisen more than three billion years ago in a high temperature environment of boiling water. Thermophilic bacteria in hot springs of Yellowstone National Park may be relict populations of the first life on earth. In fact, these thermophilic bacteria may be the ancestors of all other life forms, including humans!

Now the mixture is once again heated to 95^o C and the double-stranded DNA molecules containing the target genes separate into single strands. But now there are four single strands from two double-stranded genes. The mixture is once again cooled to 52-54^o C and the primers anneal to the strands at start positions before each gene. DNA polymerase once again catalyzes the rebuilding of each single strand into four complete double-stranded genes. PCR is called polymerase chain reaction because the reaction occurs repeatedly in cycles as duplicate copies of genes are produced exponentially. After only 40 cycles there would be 1.0995116 X 10¹² or more than one trillion copies of the original gene!

The two double-stranded DNA molecules (DNA ladders) separate into four strands. DNA polymerase will rebuild the complementary strand for each ladder, resulting in four double-stranded DNA molecules. See Animation Of Exponential Gene Replication 12. Using Lice DNA To Date The First Clothing Worn By People One of the most novel uses for DNA sequencing is the determination of when humans first began wearing clothing. According to Mark Stoneking and his colleagues at the Max Planck Institiute for Evolutionary Anthropology in Leipzig, Germany, we started wearing clothing about 70,000 years ago. This date is based on genes of human sucking lice. It correlates with the approximate time when the body louse evolved from the human head louse and corresponds to the time when the body louse's habitat (clothing) became widespread. This is also the time when Homo sapiens sapiens began moving out of Africa into cooler regions of Europe. Human sucking lice (Pediculus humanus) belong to the wingless, parasitic insect order Anoplura. [The plant genus Pedicularis is called lousewort.] Anoplurans use a set of long hypodermic-like stylets to pierce the skin and withdraw blood. After ingesting blood their body becomes swollen and shows a dark clot of blood in their abdomen. There are two forms of human sucking lice, the head louse (P. humanus capitis) and the body louse (P. humanus humanus). The head louse infests the hair of the scalp and the body louse lives in clothing near the body surface. Human lice are also known as "cooties" and their eggs attached to hairs are called "nits." Human lice cause local itching, but the discomfort is minor compared with the misery of the bacterium they can transmit called Rickettsia prowazeki. This minute bacterium causes "Epidemic Typhus," a serious disease that has devastated populations in medieval Europe. Rocky Mountain Spotted Fever is caused by Rickettsia rickettsia and is transmitted to humans by various species of ticks, most commonly the dog tick and wood ticks. The dreaded Lyme disease is caused by a spirochaete that is transmitted by the Western Black-Legged Tick (Ixodes pacificus). Stoneking and his colleagues Ralf Kittler and Manfred Kayser compared mitochondrial DNA sequences from head and body lice. The greater the difference in sequences between the two forms of lice, the older their evolutionary split. Human lice from Africa are more genetically diverse than lice from other parts of the world, indicating that the species originated in Africa. Head lice are more diverse than body lice, showing that they are the older group. By comparing the mitochondrial DNA of body lice to chimpanzee lice, Stoneking's team was able to approximate the origin of body lice to around 70,000 years ago. This date correlates well with the growing evidence that modern humans evolved in Africa and migrated northward around 100,000 years ago. Stoneking is also studying human crab lice (Pthirus pubus) which typically inhabit pubic hair. Human pubic lice are more closely related to gorilla lice than to head lice. Since this sucking louse only inhabits hairy places on the body, it might shed some light on when humans lost their heavy body hair. For More Information About The Origin Of Body Lice: Kittler, R., M. Kayser and M. Stoneking. 2003. "Molecular Evolution of Pediculus humanus and the Origin of Clothing." Current Biology 13: 1414 - 1417.

A bird louse (family Menoponidae)

There are two orders of true lice, the sucking lice (Anoplura) and the chewing lice (Mallophaga). Sucking lice have a set of long hypodermic-like stylets to pierce the skin and withdraw blood of mammals. They include head lice, body lice and crab lice. Chewing lice typically gnaw on fragments of feathers, hair and skin with a pair of mandibles. The latter lice are often called broad-headed bird lice (see image at left). Each leg is tipped with a sharp claw which can be very irritating to the host animal. The head is much broader than the human louse shown above.

Another Image Of The Broad-Headed Bird Louse

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