The Cell Terminology DNA and RNA Genetic Mutations The Cell Membrane Central Dogma Classes of Cells Cell Death Undoubtedly you have heard the phrase, “You are what you eat.” A proportion of our diet is protein and we are certainly determined by our proteins. They shape every part of our bodies, from the composition of the smallest cells to the largest organs. Proteins are composed of polypeptides, which are composed sequences of amino acids. A protein is obtained by properly folding and bending the polypeptide.
Terminology The cell is the fundamental unit of all living matter. The major characteristics that characterize living cells are: 1. 2. 3. 4. 5.
Self-feeding or nutrition: they take up chemicals from their environment, transform them, release energy, and eliminate waste products. Self-replication or growth: they can direct their own synthesis, growing and forming two cells. Differentiation: they undergo changes in form and function. Chemical signaling (chemotaxis): they respond to and sometimes produce chemical and physical stimuli in their environment. Evolution: they evolve and change via natural selection to optimize their existence.
Although this may sound like an exercise in semantics, anything that does not satisfy all five of these requirements is not a living cell. Cells are 90% water. The remaining dry ingredients are: 50% protein, 15% carbohydrate, 15% nucleic acid, 10% lipid (fat), and 10% other stuff. The approximate chemical element composition of a cell is typically: 60% hydrogen, 25% oxygen, 12% carbon, 5% nitrogen, small amounts of phosphorus and sulfur, and traces of other elements.
The Cell Membrane The cell membrane is composed of a lipid bilayer1. Each part of the bilayer is composed of structures that look like , which has glycerol and a negatively charged phosphate group at its head and two hydrocarbon chains of phospholipids along its tails. Each tail is hydrophobic, meaning that it avoids water, while the head is hydrophilic, meaning it seeks water. The water-seeking heads face the watery inside and outside of the cell and the wateravoiding tails face each other. The membrane separates the wet interior of the cell from its wet environment. The membrane is about 5 nm thick2.
An actual cell membrane has additional proteins, some of which serve as channels for the passage of various substances.
1 2
Also called a Langmuir-Blodgett layer by physicists. One nanometer (nm) is one billionth (10-9) of a meter.
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Classes of Cells There are two classes of cells: prokaryotes and eukaryotes3. Prokaryotes have no differentiated nucleus, whereas eukaryotes do have a differentiated nucleus and it is enclosed within a nuclear membrane. There are only two subclasses of prokaryotes: Bacteria and Archaea. All multicellular organisms are eukaryotes. The average eukaryotic cell is about ten times larger than the average prokaryotic cell. A eukaryote nucleus is shown below. The nucleus of a cell is not a smooth spherelike surface, as the picture below seems to indicate. In fact, it is quite convoluted, with fissures and tunnels covering its surface. The surface area to volume ratio is higher for the nucleus than it is for the cell itself4. The nuclear membrane, as shown to the left, is not a closed surface, rather it has many irregularly spaced holes. These holes prove instrumental in HIV’s ability to enter the nucleus and change the cell’s genetic machinery.
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1. Some authorities argue that there are three separate categories: Archaea, Bacteria, and Eukaryotes. They do this on the grounds that the name prokaryotes implies that eukaryotes have evolved from prokaryotes. In fact, eukaryotes are more similar to Archaea than to bacteria and the evolutionary line is not consistent. This remains controversial at this date, 10/1/2010. 4 For a perfect sphere this ratio is 4πr2/(4πr3/3) = 3/r. Therefore, the larger the sphere, the smaller the ratio. The rougher the surface, the more surface area, and the higher the numerator of this fraction.
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DNA and RNA The cells of prokaryotes and the nuclei of eukaryotes contain DNA and RNA, both nucleic acids— deoxyribonucleic acid and ribonucleic acid, respectively. These two chemical compounds are composed of purines: adenine and guanine, and pyrimidines: thymine and cytosine. But thymine is replaced by uracil in RNA. These compounds appear in complementary pairs or bases as A-T/A-U or G-C. DNA is coiled into a double-stranded helical shape, while RNA is mostly single-stranded but not coiled. The following ball-and-stick model illustrates the shape of DNA. The human genome contains some 3 billion base pairs. If we uncoiled one of our DNA molecules—of which there is one in every nucleated cell in our body—it would stretch nearly one meter! Three consecutive bases can code for an amino acid and these triples are called codons. The codons are words of a language. You need one codon for each amino acid. Most amino acids have several different codons, e.g., UUA, UUG, CUA, CUC, CUG, and CUU are all RNA codes for leucine. Exons are the coding regions and introns are the noncoding regions on DNA. The following table is a listing of the codings for those 20 amino acids that the human body needs. The codes are like sentences that tell us how to construct the proteins.
Think of the codons as words in a (short) dictionary. Then the 20 amino acid translations listed below are their definitions and this table is the unabridged dictionary for the language of protein production. Notice that there is also a code word for stop that indicates when a protein code (sentence) is complete.
Amino Acid Alanine Arginine Asparagine Aspartic Acid Cysteine Glutamic Acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalinine
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Codons for Amino Acids 3 Letter 1 Letter RNA Codons Abbreviation Abbreviation Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe
A R N D C E Q G H I L K M F
GCA, GCC, GCG, GCU CGA, CGC, CGG, CGU, AGA, AGG AAC, AAU GAC, GAG UGC, UGU GAA, GAG CAA, CAG GGA, GGC, GGG, GGU CAC, CAU AUA, AUC, AUU UUA, UUG, CUA, CUC, CUG, CUU AAA, AAG AUG UUC, UUU
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Proline Serine Threonine Tryptophan Tyrosine Valine Stop
Pro Ser Thr Trp Tyr Val
P S T W Y V .
CCA, CCC, CCG, CCU UCA, UCC, UCG, UCU, AGC, AGU ACA, ACC, ACG, ACU UGG UAC, UAU GUA, GUC, GUG, GUU UAA, UAG, UGA
As you can see, many amino acids have multiple codes, each appropriately called a synonym. All in all, there are 43 = 64 possible codings, 61 of which code for amino acids and the remaining three carry the stop code. Each cell uses these codes to make the amino acids available in its cytoplasm. A gene is any connected or disconnected segment of DNA that encodes for a specific polypeptide. Thus the gene is the set of rules for word usage that makes the language valuable for transmitting information. All proteins are composed of polypeptides, which are sequences of amino acids5 and each amino acid has the RNA coding(s) listed in the table above. Proteins can contain as few as 30 amino acids to as many as several thousands. Chromosomes are constructed from genes and there are 46 human chromosomes consisting of 23 pairs, one pair of which is either an XX or an XY chromosome (and in rare circumstances XXY, XXXY, etc., XYY, or XXYY6). In the past, noncoding regions of DNA have been referred to as “junk DNA.” Recent research has discovered that introns code for small segments of RNA that are not used in protein production. Some genes produce rather small segments of RNA—21 to 23 base pairs. These are called micro-RNA or miRNA. When initially produced as longer segments, they tend to fold over on themselves, thus creating small double-stranded RNA. These are present in many species, including about 200 of them in humans. In yeast, micro-RNA can bind to a chromosome and shut off a gene. Hence, some of these small pieces are called small interfering RNAs or siRNAs or RNAi. The latest technology is to create these RNAi and use them to turn off genes related to disease. Although research is proceeding apace, much remains to be done. To add complication to complication, each gene of the DNA helix is wrapped around a switchable long chain chemical consisting of 8 histones; together they are called chromatin. The condition of the chromatin corresponds to an on/off switch. When the switch is on, the gene is expressed, when off, the gene is not expressed. For this reason, some people with genes having a high correlation with disease do not get the disease; those genes are not switched on/expressed7. The first free-living organism to have its complete genome sequenced was Haemophilus influenzae in 1995. Since then more than 800 organisms have joined the ranks of the completely sequenced. As examples, influenza virus has 11 genes, the bacterium E. coli has 4149 genes, the simple fruit fly was found to have 14,889 genes, the chicken has 16736 genes, and grapes have 30434 genes. The study of the human genome culminated in what was purported to be the complete listing of the DNA base pairs8. One result of the study was that the human genome contains a mere 223339 genes, half as many as rice! A further surprise was that all humans share a sizable part of the basic genome 99.9%. Contrary to previous beliefs, parents do not pass down one copy of each gene. Rather, they can give their offspring as many as ten copies of a
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Over 100 amino acids are found in nature but only these 20 are needed for human growth. There are ten essential amino acids, which must be obtained from food. They are isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, which cannot be synthesized by the body. Argninine and hystidine can be synthesized, but not in sufficient amounts for growing children. Nonessential amino acids, which can be produced by the liver, are alanine, aspartic acid, arginine, citrulline, glutamic acid, glycine, hydroxyglutamic acid, hydroxyproline, norleucine, proline, and serine. 6 XXY occurs in 20 in 10,000 newborn males births, XXYY occurs in 1 in 10,000, and XYY occurs in 3–5 in 10,000. 7 Perhaps more interestingly, almost all humans have 23 pairs of chromosomes. Males have an XY-chromosome pair and females have an XX pair. Since each of the female X chromosomes contains 1098 genes that code for various proteins, there is significant overlap. If both Xs were to produce their coded proteins there would be major problems. Therefore, one of the X-chromosomes is expressed and the other is mostly (75%) turned-off, while the remaining ones are permanently activated, thus expressed at twice the level in women as in men. 8 The DNA of humans and chimpanzees is in at least 98.5%, and possibly as high as 99.4%, agreement, while that of humans and mice is in 75% agreement. The variability from human to human is less than the variability from chimp to chimp. 9 There are currently at least three groups analyzing the human genome. The RefSeq database maintained by the National Institutes of Health lists 22333 genes that encode for proteins, the Gencode project list 21671, and the Mammalian Gene Collection lists 18877.
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single gene and on the other hand they need not transmit any copies of some genes10! Researchers have found that at least 12% of the genome—that includes 2900 genes and regions between them—can differ greatly between people. The average weight of the human genome is 3.3 picograms (pg) = 3*10-12 gram. Hummingbirds average 1.03 pg and some salamanders have genomes whose mass exceeds 100 pg. As far as is known today (01/01/2011), race is not a genetic factor that distinguishes between the DNA of various people, although there is a difference in gene expression between Asian and European individuals. More importantly, your genome is not fixed for all of your life. Change is the only constant. Mutations, jumping genes, transposons, all contribute to this continuing variability. Even with that seemingly high degree of identity, there remain about three to four million base pairs at which we humans can differ11 from each other. Sidebar: Besides its many forensic uses, DNA analysis has been used to discriminate among the various breeds of dogs. Despite what the American Kennel Club says, four basic clusters emerge: (1) the oldest dogs, (2) guarding dogs, (3) herding dogs, and (4) hunting dogs. Some of the breeds in each cluster are shown below. Oldest Alaskan malamute Chow chow Pekingese Shar-pei Siberian husky
Guarding Boxer Bulldog German shepherd Mastiff Rottweiler
Herding Belgian sheepdog Collie Old English sheepdog Saint Bernard Shetland sheepdog
Hunting Airdale terrier Beagle Bloodhound Golden retriever Pointer
Central Dogma of Biology The Central Dogma of Biology states that for all living cells:
DNA → RNA12 →
protein synthesis
The first step () is called transcription and the second () is translation. In transcription, the coding on DNA is transcribed onto mRNA (messenger RNA). This occurs by matching base pairs on one strand of the DNA to the mRNA as shown in the following figure. The mRNA then binds to a ribosome, where the proteins will be made.
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This is called copy number variation. Identical twins frequently have different copy numbers in several genes. Usually the copy number ranges from zero to five, but in rare cases it can range from 5 to 368. 11 Prior to the completion of the Human Genome Project, the philosophy of Genetic Determinism held sway. It claims that the genome with which you are born completely determines you, from physical types, disease susceptibility, all the way to sexual preference. But with so few genes, this theory no longer commands the attention it once did. In fact, randomness associated with the many linkages between genes gives rise to a whole host of other, as yet unaccounted-for, factors. 12 Research published in 11/2010 showed that in white blood cells from 27 different people, on average, as many as 4000 genes had misspellings in their RNA that was not in their DNA. In fact, RNA contained misspellings at more than 20000 different places along their genome and about 10000 misspellings in two or more of the people studied. Why and how these occur is, at present, unknown.
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Transcription from DNA to RNA
Transfer RNA is a small molecule consisting of only 80 nucleotides, containing anticodons13. Its function is to deliver amino acids to the polypeptide molecules being made in the ribosome. The sequence of nucleotides forming DNA dictates the sequence of amino acids needed for constructing the protein. During translation, each section of tRNA links to its appropriate free-floating amino acid within the cell. These amino acids are then carried to a ribosome. There the tRNA bonds with mRNA that has attached to the ribosome. Then, codon by codon, a chain of amino acids is assembled into a protein. The Central Dogma of Biology holds for all life forms found to date on this planet14, subject to the transcription errors noted in footnote 12. The very complex molecular structure of a ribosome is shown below.
Ribosome
Just as DNA is the master recipe for proteins, mRNA is the user’s recipe, tRNA is the collector of the ingredients, and the ribosome is the “chef” or “kitchen,” where the language of the words coded for in DNA and RNA is used to construct the proteins out of which all living cells are made. A protein is a rather complex and heavy molecule15. The following is a computer-generated image of the protein with the coded name p21 H-ras. The p21 means that it is a protein with a molecular weight of 21 KDaltons = 21,000 Daltons.
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The anticodon to UAC would be AUG. This is where that semantic difference for the definition of a living cell enters. Viruses do not satisfy all five requirements of a living cell. Hence, they are not considered to be living and they need not follow the Central Dogma of Biology. But, more of this later. 15 During conditions of starvation (and diabetes mellitus), the body draws from its amino acid pool, and not its fat stores, to make glucose. So, long-term fasts are not a medically wise way to lose weight. 14
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Cell Death Considering that eukaryotic cells reproduce by mitosis, doubling each cycle (the average adult human produces (and kills) about one billion = 1,000,000,000 = 109 cells per day), if not for cell death it wouldn’t be long before there were more cells than an organism could possibly sustain. Also, cells age and wear out; some take just a few hours and others can last for decades, depending on the stressors encountered16. For this reason all cells carry an inherent coding called programmed cell death. After a cell has split a certain number of times, it dies. Without the presence of this mechanism, cell proliferation would destroy the organism. In humans, our skin cells die and slough off at a regular rate. After a shower, take a towel and rub your arm; what you’ll see is a many celled mixture of cells in the dead skin that is exfoliating. There are four known forms of programmed cell death, but we will only concern ourselves with one of those. Apoptosis is triggered by the enzyme caspase. The cell membrane softens, the nucleus shrinks and divides, and the entire cell starts to bleb or balloon and oscillate. In short order, the cell lyses or bursts. The picture below shows a cell beginning to bleb.
A cell beginning to bleb prior to lysis.
Genetic Mutations The genomes of many organisms are far from immutable, some change slowly and others change rapidly. The smallpox virus seems to have remained unchanged for many centuries, if not millennia. The influenza virus’s genome can change during the course of a single flu season, using antigenic drift and/or shift. As we will see, HIV has a genome that is very likely to mutate in a relatively short time. How can such mutations affect the rest of the organism? In the following, we will look at some human mutations. As a specific example, research discovered a genetic mutation that caused the production of steroid 5areductase. This chemical is responsible for the transformation of testosterone to dihydrotestosterone, of which the latter is 50 times as potent as the former. In one codon, adenine (A) has been replaced by a (G) guanine. This replacement results in the replacement of alanine in place of threonine (Check the codon table.) in the finished protein. The end result of this single mutation is a five-fold increase in the risk of prostate cancer. Sickle cell anemia is an inherited disease where a single amino acid substitution (valine replaces glutamic acid) has profound effects. This alters the production of red blood cells, changing them from disk-shaped to crescent or sickle-shaped. People who are homozygous—having that mutation on both chromosomes—have about 85–95% of 16
Noncancer cells usually obey the Hayflick limit, whereby they replicate about 50 times and then die.
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their red blood cells affected. This leads to severe anemia leading to episodes of severe pain and other serious symptoms due to the low oxygen levels in various parts of their body. Consequently, their life expectancy is considerably shortened. People who are heterozygous—having that mutation on only one of their chromosomes— have about 40–45% of their red blood cells affected. They rarely suffer to the extent of the homozygous. Both forms of the disease result in resistance to infection by the malarial parasite, indicating its likely origin in the malarial areas in Africa. You may have heard of the breast cancer genes: BRCA1 and BRCA2. A woman with a specific mutation on BRCA1 was thought to have at least a 60% higher lifetime risk of developing breast cancer. In the US, that probability is 0.13, so that the mutation would lead to a probability of 0.21. Besides that, there is a gene CHEK2, with a fairly low mutation rate. But, if you have the mutated CHEK2, a woman’s risk doubles and a man’s risk17 increases by a factor of ten! Defects in genes on chromosome 17 have been analyzed in some detail. The following table lists some of those genes and their corresponding medical condition. Gene Rp13 CTAA2 MYO15 COL1A1 NF1 SGCA BRCA1 GH1 SSTR2
Medical Condition Retinitis pigmentosa18 Cataract Deafness Osteogenesis imperfecta19 Neurofibromatosis20 Muscular dystrophy Breast and ovarian cancer Growth hormone deficiency Small cell lung cancer
The gene P53 acts as a tumor suppressor. Hence, any mutations it might experience usually result in the growth of cancer cells21. Fully half of all cancers are thought to be associated with mutations in this particular gene. Furthermore, there is research that shows that all cancers are the result of a small number of mutations in a set of fewer than 100 genes. On January 1, 2003, the complete coding of human chromosome 14 was announced. Only human chromosomes 22, 21, and 20 had been previously analyzed in full. Chromosome 14 is the longest human chromosome consisting of 87,410,661 nucleotides, constituting about 3% of the entire human genome. It contains 500 known genes and about 350 suspected genes. It also is home to a gene, neurexin 3, which consists of about 1.7 million base pairs. This gene is thought to be associated with the construction of synaptic connections in the brain. Mutations on this chromosome are responsible for over 60 diseases, ranging from achromatopsia (complete color blindness) to Usher Syndrome. One of this gene’s mutations may cause early onset Alzheimer’s disease. Some other conditions known to be associated with one or more genetic defects on other chromosomes are: basal cell carcinoma, colorectal cancer, cystic fibrosis, diabetes mellitus, dyslexia, fructose intolerance, hemolytic anemia, Huntington’s disease, leukemia, muscular dystrophy, progeria, schizophrenia, severe obesity, Tay-Sachs disease, and Tourette’s syndrome. And the list goes on and on. The average human carries from 250 to 300 defective copies of genes and 75 of these variants are associated with disease. Many factors can impinge on the DNA present in the nucleus of human cells, e.g., ultraviolet light, ionizing radiation, and reactions with teratogenic22 chemicals are just a few. The developing fetus is partially protected by the placenta. This provides a barrier to most molecules of weight in excess of 1000 Daltons. Unfortunately, there are very many lower molecular weight chemicals and drugs from which there is no protection, most notably alcohol. The Food and Drug Administration, FDA, has categorized many drugs by their possible effects on a developing fetus as follows: 17
Yes, men do get breast cancer. In the US that rate is 0.01, while in Turkey, it is much higher. This disease begins in early childhood when the retina begins to deteriorate, usually leading to blindness. 19 A bone disease characterized by malformed bone matrix with normal calcification. There is a tendency to frequent bone fractures with normal healing. In some cases, this tendency of the bone to fracture decreases and may disappear completely. 20 This disorder affects cell growth of neural (nerve and brain) tissue. 21 Cancer cells have an impaired programmed cell death function, so they grow unabated—as if they are immortal. 22 The word means causing embryonic or fetal mutation. 18
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A. B.
C.
D.
X.
Controlled Studies Show No Risk. Adequate, well-controlled studies in pregnant women have failed to demonstrate a risk to the fetus in any trimester of pregnancy. No Evidence of Risk in Humans. Adequate, well-controlled studies in pregnant women have not shown increased risk of fetal abnormalities despite adverse findings in animals, or, in the absence of adequate human studies, animal studies show no fetal risk. The chance of fetal harm is remote, but remains a possibility. Risk Cannot Be Ruled Out. Adequate, well-controlled human studies are lacking, and animal studies have shown a risk to the fetus or are lacking as well. There is a chance of fetal harm if the drug is administered during pregnancy; but the potential benefits may outweigh the potential risk. Positive Evidence of Risk. Studies in humans, or investigational or post-marketing data, have demonstrated fetal risk. Nevertheless, potential benefits from the use of the drug may outweigh the potential risk. For example, the drug may be acceptable if needed in a life-threatening situation or serious disease for which safer drugs cannot be used or are ineffective. Contraindicated in Pregnancy. Studies in animals or humans, or investigational or post-marketing reports, have demonstrated positive evidence of fetal abnormalities or risk which clearly outweighs any possible benefit to the patient.
Some examples of common drugs that pose a high risk, as categorized above, include: • D: lithium (antidepressant), paclitaxel (anticancer), phenobarbital (anticonvulsant); • X: lovastatin (widely used for lowering blood cholesterol), accutane & tazarotene (widely used in treating severe acne and psoriasis), and warfarin (one of the most commonly used blood thinners). Surprisingly, not all FDA approved drugs have been assigned a pregnancy effect category!
References Klug & Cummings – Essentials of Genetics, 5th edition Perta & Salzberg – Genome Biology (2010)
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