GENETICS

Prof Atsma 2004

Introduction

The study of the transfer of hereditary material from parent(s) to offspring is the science of Genetics. For asexual reproduction, this would be extremely predictable, since offspring are typically exact copies of the parent.

Sexual reproduction is arguably one of the greatest inventions of biological evolution. An organism would otherwise be totally dependent on the snail's pace accumulation of mutations for evolutionary change. Mixing of DNA from two different individuals allows for more variation than asexual reproduction. Mutations/new variations can be tried out in numerous ways throughout the species. However, a new form of cell division was necessary for sexual reproduction to become possible.

Meiosis

Similar in its basic mechanics to mitosis, meiosis allows for both cell division and the halving of the amount of DNA in the sex cells or gametes produced. See your textbook for diagrams so that you may compare mitosis and mitosis. The overall difference between the two is that mitosis doubles the chromosomes once and divides once (leaving two cells identical to the original cell); Meiosis doubles the chromosomes once, but divides twice (leaving two cells with half the chromosomes of the original cell). The need for this is obvious when you recall that the whole point of sexual reproduction is to combine cells from two organisms, while ensuring that the offspring will have the same number of chromosomes as the original parent cells.

OK, so we understand that the extra division is the overall strategy that makes meiosis so different. The specific step that makes the precise halving of chromosomes possible occurs in "Metaphase I" of meiosis. As you review the book's diagrams, note that the chromosomes line up "two-by-two" in this stage, while they line up along the metaphase plate in single file during regular mitosis. This assures that one of every chromosome will end up in each of the four gamete cells at the end of meiosis.

Meiosis makes the study of reproduction and inheritance infinitely more complicated than it was for the first several million years of life on Earth. Barring mutation, a life form going through asexual reproduction based upon mitosis would be expected to look exactly like its parent. Depending upon the number of chromosomes in the cells of the life form, and how much variation there is within the species, there can be thousands or millions of different possible offspring from each sexual reproduction event. The genetics of sexually reproducing organisms can be one of the most fascinating topics in biology.

PRELUDE TO THE SCIENCE OF GENETICS

The idea of inheritance of traits has probably fascinated people since the beginning of civilization. But without a clear conception of the importance of the scientific method, or any idea of how cells worked (never mind the comparatively advanced level of biology you now understand), even the greatest minds of the 1800's concocted very poor hypotheses regarding why family members look alike. The homunculus "theory" suggested that the sperm was actually a little person who grew inside the female. Pretty silly idea, isn't it, especially since it doesn't explain how offspring could resemble their mother instead of their father. To solve this, a school of thought, loosely referred to as the humoral "theory" suggested that the "vital" fluids responsible for life were distilled in the sperm and egg. The mixing or blending of the two would cause the fluids to gel into a little person who would gradually grow into a baby. Perhaps a plausible solution to how action figure toys got here, but this is hardly any kind of specific biological explanation.

MENDELIAN GENETICS

The Monk Who Flunked

Amid this historic backdrop in the mid 1800's, a Monk named Gregor Mendel studied mathematics and science. The story supposedly goes that he did not pass the University tests required to become a teacher, so he returned to the monastery. The Abbot allotted Mendel a respectably-sized garden so that he could do a little research. In this garden, pea plants and the scientific method flourished, and the science of Genetics was born.

MENDEL AND THE SCIENTIFIC METHOD

Although evidence and some critical speculation may suggest that his experiments were far from perfect in design or execution, it is clear that Mendel's work was probably one of the earliest examples of use of the scientific method. First, he used observation and determined that pea plants had several clearly identifiable traits (such as flower color, pea shape, size of plant, etc.). He then wondered if there was a measurable pattern to the inheritance of these traits. He formed a generalization and proposed questions about it based upon his observations that pure-bred plants would produce offspring exactly like themselves through numerous generations. This seemed true - pure-bred plants with purple flowers never produced offspring with white flowers and visa versa. This "revelation" alone was of little value to science (farmers may have already known at least this much), but his next step is what set Mendel on the road to some important discoveries. He decided to collect observations about a cross between opposite pure-bred strains.

THE MENDELIAN "LAWS"

Crossing (plants don't "mate", they cross-pollinate) a purple-flowered and white-flowered plant, or a tall and short ("dwarf") plant was a simple yet effective test for the "blending inheritance" idea kicking around in Mendel's day. Yet he did not get pink flowered or medium-sized offspring. All offspring of such pure-bred crosses had purple flowers and were tall. From this he formed the hypothesis, later incorrectly referred to as the "Law" of Dominance and Recessiveness. Apparently, whatever was responsible for causing these traits (unlike you, he did not know about DNA, chromosomes, and meiosis) did not simply blend together, but could have the ability to completely overshadow (dominate) the other trait (the recessive trait). Again, if he would have stopped there, his work would have been somewhat worthwhile, but very unremarkable.

But he didn't stop there - he crossed these offspring among themselves (crossing of sisters/brothers is not illegal in plants). Again, he did not get blending (no pink flowers or medium-sized plants), but he did get a few white flowers and a few short plants. Whatever was responsible for causing these traits did not blend, stayed separated, and also did not become lost or assimilated in the dominant trait. He referred to this as the Law of Segregation.

"FACTORS" AND THE PUNNETT SQUARE

Again, Mendel was working in the dark (figuratively speaking), and did not know that "genes" (small sections of a chromosome) were responsible for these traits. But he did not let that stop him. He correctly reasoned at some level that it might be somebody else's job (perhaps Watson and Crick, who were not born yet) to figure out what part of the cell would be responsible for trait transmission. So, he called the "things" responsible for producing traits "factors." Now he had something to work with mathematically! After reviewing his data, he reasoned that there must be two factors for each trait in any organism, but that only one of these pairs is passed on from each parent (review meiosis and see if he was correct). He further reasoned that only one dominant factor is necessary for the offspring to be just like the dominant parent (with regard to that particular trait).

A Punnett square is a visual demonstration of simple genetic probabilities that doesn't work nearly as well for some complicated genetic crosses. It is a simple graphical device where each parent's "factors" or gene varieties that can end up in the gametes produced are placed along the top and side of a box. The box is divided into columns and rows depending upon the number of possible gametes for each trait to be studied. See the first punnett square for a simple example of crossing a pure-bred tall plant with a short one. Notice that the first generation is all "Tt," having one tall and one short factor. Thus, if you recall the "Law" of Dominance and Recessiveness, all of the offspring should be tall, even if they carry the short factor along for the ride. Recall that for Mendel's second cross among these offspring, you would use these "Tt" offspring as the parents of the next generation. This can be demonstrated with another Punnett square. Note that three out of the four boxes have at least one capital "T," and would represent tall plants. One box alone has two lower case "t's" and would represent the recessive, small variety. This 3:1 ratio in the second generation is the same as Mendel's 3/4 tall to 1/4 small data.

There is a 3:1 ratio of tall to short plants. (The presence of even one capital T=a tall plant.)

But Mendel had one more question to ask and one more set of observations to explain.

THE "LAW" OF INDEPENDENT ASSORTMENT

What if we wish to follow two traits through several generations - such as tall, purple-flowered plants crossed with short, white-flowered plants. From the above, you would expect the first generation to be all tall and purple-flowered (if you did not expect that, re-read the "Law" of Dominance). But what of the second generation? One might predict that 3/4 would be tall/purple, and 1/4 short/white. But that is not what happened. Yes, he did get some tall/purple and short/white offspring, but he also got a significant number of "mixed" offspring (tall/white & short/purple). Mendel reasoned that in this "dihybrid cross," the factors were not only separate (or segregated) from their matching dominant factor, but also separate from other factors too. Thus, when the gametes are produced to begin with, the tall factor was not stuck with the purple factor, but was free to (randomly) travel with either the purple or white flower factor. Later more properly renamed, this "Law" became known as the Principle of Independent Assortment. The Punnett square pictured below demonstrates this dihybrid cross and the different possible gametes from either the mother or father plant.

Note that there is a 9:3:3:1 ratio of Tall/Purple (9): Tall/white (3): short/Purple (3): short/white (1). This is exactly the same phenotypic ratio that you should see in all dihybrid crosses that follow the Principle of Independent Assortment.

PROBABILITIES

Although Punnett squares are great ways of visually demonstrating the possible outcomes of a cross, probabilities are often better ways of determining results for more complicated crosses. Probabilities are expressed as a fraction of one (the "whole" pie in a pie chart). The simple rule as it applies to genetics is that probabilities of events that must happen simultaneously (like mixing of egg and sperm in mating/crossing) are multiplied. Non-simultaneous events and alternate probabilities are added.

For a simple example, let us use the tall/short F1 monohybrid cross (Tt X Tt). What are the odds of obtaining a "TT" offspring? The odds of one parent giving only its capital "T" would obviously be ½ or 0.5. Since each parent would have to simultaneously give its tall allele, we would multiply ½ X ½. Checking back to the Punnett square you will see that this result (½ X ½ = 1/4) matches the one out of four boxes that were "TT".

For a more complex example, let us check the probability of obtaining a short purple-flowered plant from our dihybrid cross of TtPp X TtPp. Both parents must donate their short (t) allele, but either one or both can be the source of the purple (P) allele(s). In short, there are three ways for the parents to donate gametes in order to get a short purple-flowered plant: tP/tp=ttPp; tp/tP=ttpP; and tP/tP=ttPP.

Each of these events has a 1/16 probability: As already discussed, there is obviously a "50-50" (or ½) chance that each parent will donate a particular allele. So, for the tP/tp possibility mentioned in the previous paragraph, parent "A" has a ½ chance of donating the t allele, and a ½ chance of donating the purple allele (½ X ½ = 1/4). Parent B has the same odds of "choosing" its t and p alleles (again, ½ X ½ = 1/4). So for this possibility, 1/4 X 1/4 = 1/16. But since there are two other ways to get the same phenotype we must add the two other possibilities as well (1/16 + 1/16 + 1/16 = 3/16). Compare 3/16 to the Punnett square done earlier for this dihybrid cross. How many of the 16 boxes were short/purple?

MENDEL UPDATED

So far, we have attempted to present this from Mendel's perspective since it is a valuable lesson in the scientific method. We should mention here in all honesty that some say Mendel did fake and/or ignore some of his data once he saw the patterns developing, which would if true be rather poor use of the scientific method. Since there is little if any evidence that Mendel did anything significantly dishonest, one may prefer to be a little bit more charitable and suggest that pioneers in any area are certain to make mistakes or encounter things they cannot explain. At any rate, Mendel’s work went unnoticed for decades until biologists working on their own primitive experiments in heredity discovered his writings.

Over the hundred or so years since rediscovery of Mendel’s work, we have learned where he was right and wrong. His "laws" apply to pea plants and many other species on earth, but not to all. The science of cell biology has grown tremendously as well.

Since we know about chromosomes and genes (Mendel's "factors"), we should bring our discussion up to 20th century vocabulary. The phenotype is the observable trait actually expressed ("tall" or "short"). Each variety of a gene for particular trait is called an allele. For Levi’s genes, or jeans, there are regular and "501" jeans. Figuratively speaking, both Levi’s jeans could be considered alleles of the lower body covering jean. For more serious examples - for the size trait in pea plants, there are tall and short alleles; for the flower color trait, there are purple and white alleles, and so on. Specifically, "T" is the tall allele, and "t" is the short allele, etc.

Since we bring these alleles together to form a single cell or "zygote," the suffix zygotic is used to describe the genotype or actual alleles present in the offspring. When describing genotype in words (not letters as in "TT," "Tt," or "tt"), the terms "homozygous" or "heterozygous" are used to describe pure-bred and mixed alleles respectively. Examples: TT = homozygous tall; Tt = heterozygous tall; tt = homozygous short. The original pure-bred parents are simply referred to as the Parental or "P" generation. Their offspring are called the "F1" generation, and the offspring of these hybrids are called the "F2" generation ("F" is for "familial").

BEYOND MENDEL

As significant and precocious as Mendel's work was, the truth is that there are numerous exceptions to Mendelian Genetics. Mendel's "Laws" work well for pea plants and for millions of traits that can be studied in living things. But they do not hold up for many other organisms and their traits. Following are selected topics that go beyond Mendel’s principles in explaining patterns of heredity.

INCOMPLETE DOMINANCE

Mendel demonstrated that the alleles remain segregated, but that doesn’t mean that the phenotype can’t ever be a mixing or blending of the products of two alleles. A cross between red and white four-o’clock flowers produces offspring with pink flowers. A cross of this hybrid generation produces a 1:2:1 ratio of red to pink to white flowers (do the punnett square of Rr X Rr if you are having trouble visualizing this).

CO-DOMINANCE

There are some situations where both alleles present can be expressed at the same time. Human blood typing is a good example. If you are blood type AB, your red blood cells produce both the A and B surface marker molecules. You are type "O" if you produce neither marker molecule. See the examples of blood type crosses below.

POLYGENETIC INHERITANCE

It is not always true that only one pair of factors is responsible for a particular trait. This was discovered in wheat, when crossing dark red and white varieties did produce all "light red" offspring. At first, researchers thought that they had simply discovered another case of incomplete dominance. However, a cross of the F1 hybrid generation produced a wide spectrum of colors: dark red, medium red, light red, pink, and white. The ratio of these phenotypes suggested the simple solution to this riddle - two pairs of alleles were responsible for color in wheat!

The parents would be stated genotypically as RRRR X rrrr. The F1 hybrids would all be RrRr. Note that there are two chromosome pairs involved and regular "R’s" are used to designate the alleles from one chromosome while the "R’s" in italics and script font are the alleles from the other chromosome. The Punnett square would be handled just like a dihybrid cross:

The ratio of phenotypes is vastly different from that of a dihybrid cross. There is a 1:4:6:4:1 ratio of Dark Red (1): Red (4): Light Red (6): Pink (4): White (1). This is phenotypic ratio is typical of polygenetic inheritance where two chromosomes carry alleles for the same trait. However, sometimes there are cases of polygenetic inheritance where three or more pairs of alleles are involved (skin color, intelligence, etc.) and a wide range of phenotypes are possible. Notice that polygenetic inheritance tends to produce far more intermediate phenotypes than those at the extremes - something that may be useful in an evolutionary context.

SEX DETERMINATION

A different kind of inheritance pattern involves the only mismatched chromosome pair in sexually reproducing organisms, namely the X & Y chromosomes. The Y-chromosome apparently carries the genes needed to produce a male, and is smaller than its partner. A person with two X-chromosomes is female, and an XY-combination is male.

SEX LINKAGE

Thomas H. Morgan, a geneticist from the early part of this century, chose the fruit fly for genetic research. The flies generated research data more quickly than plants since they reproduced in a few weeks (not once or twice a year). He used basically the same ideas as Mendel and initially showed Mendelian Genetics to work for animals as well. Until he got some odd data.

Morgan crossed a Red-eyed female fly with a white-eyed male. He got all red-eyed offspring which suggests that the principle of dominance and recessiveness is working here. Initially, the cross of the F1 generation seemed to produce the standard 3:1 ratio of red to white-eyed flies. But upon closer examination, this distribution seemed to sort itself out rather strangely between the males and females. All of the females had red eyes, but the males were split 50-50 between red and white eyes. What could explain this?

Morgan reasoned that the genes for eye color must be attached ("linked") to the X-chromosome, and that there would be no corresponding gene on the truncated Y-chromosome. All of the F1 flies are phenotypically red-eyed, but the genotypes are very different. Both females are heterozygous red eyed, but the males are hemizygous red-eyed. The term hemizygous means that they only have half the genetic representation of the female (an allele on the X, but not the Y-chromosome).

If we next cross the F1 offspring, we see that Morgan’s hypothesis fits the data. The F2 generation has all red-eyed females (even though one is homozygous and the other is heterozygous), and the males are split: 1 Hemizygous red: 1 Hemizygous white.

LINKAGE

Morgan went on to discover that linkage works for a multitude of traits, and found that the Principle of Independent assortment is in effect far less frequently than Mendel and his followers would have suspected. Many times, when attempting a dihybrid cross with his fruit flies, he would get a 3:1 ratio rather than the 9:3:3:1 ratio expected for independently assorting two-trait crosses. He applied the same reasoning used for sex linkage to explain this as well. He essentially said, "What if the sets of alleles for the two different traits are on the same chromosome?" They would not shuffle randomly (independently) into gametes during meiosis, but would be handcuffed together (again, "linked") during meiosis.

In the following example, a two-trait cross of flies with the dominant traits of dark bodies and long wings (BBLL) and flies with "albino" or white bodies with short wings (bbll), the F1 generation would be all Dark/Long wings. But the F2 might be expected to fit the 9:3:3:1 ratio of a dihybrid cross. Instead, a 3:1 ratio was obtained. Why? Well, the meiosis of the BbLl hybrids does not produce four possible gametes, but only two. In the following figure, the boldface B & L alleles are linked together, as are the italicized b & l alleles.

Only two possible gametes from the linked genes for body color and wing length.

Even though there are two traits involved, the fact that they are linked together (as shown in figure 1) means that there are only two possible gametes for each parent rather than four as in a typical dihybrid cross. Compare the Punnett square below to that of the dihybrid cross performed earlier.

The most concise way to put this is that the two traits are behaving like a single trait cross (note the 3:1 ratio) because both traits travel on the same chromosome, so there is no other chromosome involved with which to assort independently.

CROSSING OVER

Occasionally, but too often to attribute to mutation, Morgan saw some strange varieties during his linkage experiments. For example in the previous cross, nearly all of the offspring were either Dark/Long wings or white/short wings, but 1-2% would turn up as "mutts" (that is Dark/short wings or white/Long wings). This was eventually explained as an exchange of information between chromosomes during meiosis - sort of a swap of alleles. In the heterozygote, the chromosome with the Dark ("B") allele might occasionally swap its linked Long-winged allele with its neighbor’s short-winged allele. So, perhaps one in one-hundred times, you could end up with a Bl gamete instead of a BL. One in one-hundred times (or less) that could change the outcome of the previous Punnett square and produce an occasional "mixed breed."

HUMAN GENETICS AND PEDIGREE CHARTS

Since it is unethical to use humans as test subjects, and it would take too long to get results anyway, one of the best ways to study human patterns of inheritance is to go back in time (history, not time travel). A pedigree chart or "family tree" is the tool most often used. By using boxes, circles and connecting lines, it is possible to diagram a family history of a genetic disease. An example appears below.

A pedigree chart. Circles=females; squares=males; solid objects=diseased individuals; half-solid objects=carriers; unfilled objects=genetically healthy individuals. This pedigree chart is typical of X-linked disorders that often skip generations (although female carriers may be present) and appear mainly in males.

GENETIC DISORDERS

A mutation is any change in the genetic material (DNA). They fall into two main categories: nucleotide and chromosomal.

Nucleotide mutations usually affect a single nucleotide by causing the replacement of a nucleotide with the wrong one (a point mutation). These mutations typically only affect one potential codon, and rarely damage the cell or individual. But if by bad luck, the mutated point was a pivotal codon for an amino acid critical to the proper folding of the protein coded for by DNA, the consequences can be devastating (as in sickle cell anemia & hemophilia). If a nucleotide is added or deleted (a frame-shift mutation) the entire code is typically destroyed. By analogy, a point mutation is perhaps one "typo" in a whole page of text - not a big deal unless it is a key word (like the version of the Bible printed in the 1800's with the phrase "thou shalt commit adultery."). By contrast, a frame-shift mutation changes a sentence like this, to a senptenc elik ethi s (a "p" was added to the middle of the word "sentence" which then threw off the rest of the words).

Chromosomal mutations involve breaking or inappropriate attachment of chromosomes. Inversions occur when a piece of a chromosome breaks, flips, and reattaches. Duplications and deletions are self-explanatory. Non-disjunction is probably one of the most significant from the point of view of human genetics. If chromosomes become sticky during metaphase I of meiosis (as they tend to in women past the age of 35), they may fail to separate. This can leave some gametes with either an extra chromosome, or no chromosome. Although this may simply be lethal to the gamete or embryo, it may result in abnormal offspring (as occurs in Downs & Kleinfelter's syndromes).

A punnett square demonstrating the effect of non-disjunction of the sex chromosomes during the production of the female oocyte.

Kleinfelter's (XXY) and Turner's (X-) syndromes are relatively mild genetic diseases. The XXX syndrome (no, not a propensity to watch adult films) is sometimes termed the superfemale syndrome (really!), and produces a relatively normal female. Since the X-chromosome does carry some genes important for survival, the Y- genotype is lethal.

REGULATION

So far, we have generally referred to genes as parts of DNA molecules responsible for making proteins. Although the details of gene regulation go well beyond the scope of this course, it would be wrong to finish the topic of Genetics without mentioning regulatory genes.

Despite coming from the same fertilized egg as stomach cells, brain cells do not make HCl (good thing too, huh?). But why not? Brain cells and stomach cells have the same DNA. Clearly there is a way to turn genes on and off (sometimes for a lifetime). As you might guess by now, the answer is a "simple" matter of biochemistry.

Research has shown that some genes do not code for proteins or RNA at all, but serve as attachment points for regulatory molecules. Others serve as starting places for RNA-polymerase to attach. A regulatory molecule may shut off the production of a protein by attaching to DNA and blocking the ability of RNA-polymerase to roll down and copy the DNA. Conversely, stimulatory molecules may help to remove these roadblocks. This is the operon concept, which was first worked out in prokaryotic cells. Although eukaryotic cells are obviously far mor complex, it is fair to assume that similar processes turn on or shut off genes in all cells.

Although the above explanation was intentionally very brief, it may help you to understand how hormones, drugs, and other communication molecules generally work. By attaching to regulatory genes, or telling the cell to make regulatory molecules which do so, these messenger molecules may direct the activity of cells.

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When one thinks about how much of our biochemistry, reproduction, and survival hangs on behavior of these tiny threads of DNA, it is astounding that things go right as often as they do. To paraphrase a rather precocious philosopher: It is miraculous that a harp with so many strings goes out of tune so infrequently.

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