Genetics

Mendel's Law of Independent Assortment The principles that govern heredity were discovered by a monk named Gregor Mendel in the 1860's. One of these principles, now called Mendel's law of segregation, states that the alleles for a trait separate when gametes are formed. These allele pairs are then randomly united at fertilization. Mendel arrived at this conclusion by performing monohybrid crosses. These were cross-pollination experiments with pea plants that differed in one trait, for example pod color. Mendel began to wonder what would happen if he studied plants that differed in two traits. Would both traits be transmitted to the offspring together or would one trait be transmitted independently of the other? From his experiments Mendel developed the principle now known as Mendel's law of independent assortment. Mendel's Law of Independent Assortment Mendel performed dihybrid crosses (mating of parent plants that differ in two traits) in plants that were true-breeding for two traits. For example, a plant that had green pod color and yellow seed color was cross-pollinated with a plant that had yellow pod color and green seeds. In this cross, the traits for green pod color (GG) and yellow seed color (YY) are dominant. Yellow pod color (gg) and green seed color (yy) are recessive. The resulting offspring or F1 generation were all heterozygous for green pod color and yellow seeds (GgYy). Fig A

Mendel then allowed all of the F1 plants to self-pollinate. He referred to these offspring as the F2 generation. Mendel noticed a 9:3:3:1 ratio. About 9 of the F2 plants had green pods and yellow seeds, 3 had green pods and green seeds, 3 had yellow pods and yellow seeds and 1 had a yellow pod and green seeds.

Fig B

Mendel performed similar experiments focusing on several other traits like seed color and seed shape, pod color and pod shape, and flower position and stem length. He noticed the same ratios in each case. From these experiments Mendel formulated what is now known as Mendel's law of independent assortment. This law states that allele pairs separate independently during the formation of gametes. Therefore, traits are transmitted to offspring independently of one another. Genotype and Phenotype In Mendel's experiment with pod color and seed color (Figure A) we see that the genotype or genetic makeup of the F1 plants is GgYy. The phenotypes or expressed physical traits are green pod color and yellow seed color. Both of these traits are dominant.

The F2 generation pea plants (Figure B) show two different phenotypes for each trait. Pod color is either green or yellow and seed color is either yellow or green. There are nine different genotypes: F2 Genotypes------------------------------F2 Phenotypes GGYY, GGYy, GgYY, GgYy --------Green pod, Yellow seeds GGyy, Ggyy---------------------------Green pod, Green seeds ggYY, ggYy----------------------------Yellow pod, Yellow seeds ggyy --------------------------------------Yellow pod, Green seeds Test cross Mendel devised a system of conducting verification for the results obtained by him. It is known as test cross. It is a cross between F1 plant and the recessive parent. A test crossconducted for the monohybrid inheritance results in the two opposite characters expressing in a ratio of 1:1. Similarly, a test cross-conducted for the dihybrid inheritance results in the expression of the two parental combinations and the two recombinations appear in the ratio 1:1:1:1.

Significance of Test Cross

Test cross can be used to determine the genotype of the F1 plant. The test cross can be used to support the idea that the reappearance of the recessive character in the F2 generation is due to the heterozygous condition of the F1 plant.

The test can be used to verify whether any given pair of characters can be alleles (contrasting characters).

Back Cross
If an F1 individual or an individual of F2 or F3 generations is crossed with any one of the parents it is called a back cross.

Multiple Alleles The term multiple allele is a condition where more than two genes occupy the same locus, on the same pair of homologous chromosomes, in different organisms. Each of these genes expresses a totally different character. The inheritance of A B O blood groups in man is an example of multiple alleles. The four blood groups A, B, AB and O are due to the presence of three genes occupying the same locus on the same pair of chromosomes in different human beings. The discovery of blood groups dates back to the year 1900. A German doctor by name Carl Landsteiner discovered the four blood groups in man. He was examining the reason for the instant death of many persons immediately after a blood transfusion. He isolated the plasma and RBC from the blood samples of different persons. He found that whenever the plasma was mixed with the RBC of the same person, the mixture was smooth. However, when the plasma and RBC belonged to different persons, the mixture was found to be smooth in some cases and clumped in others. Detailed analysis conducted by Landsteiner showed that the human blood contained two specific substances called antigens, which are responsible for either smooth mixing or clumping. He named these antigens as antigen A and antigen B. Based on the presence or absence of these antigens, he classified the human blood into four groups namely A, B, AB and O. The following table represents the antigens and the corresponding antibodies found in the four blood groups of man. Blood Group----- Antigen in RBC--------Antibody in plasma A----------only antigen A -------------------Antibody-b B-----------only antigen in B------------Antibody-a AB------ Both antigens A and B ---------------------------Nil O------- Neither-----------------------------------Both antibody a and b

Blood Transfusion The transfer of blood from one person to another is called blood transfusion. In all cases of blood transfusion, it is necessary to match the blood group of the recipient with the blood group of donor. The following table represents the blood group matching.

From the table it is clear that persons with blood group AB can receive blood from any other person. Hence they are commonly described as universal recipients persons with blood group O can donate blood to any other person. Hence, they are commonly described as universal donors. Blood groups............ Genotypes A ........ IAIb or IAIO B ......... Ibb or IbIO AB ...... IAIb O ....... IOIO

The fruit-fly, Drosphila melanogaster has 15 alleles for eye colour. In rabbits, there are 4 alleles for colour. In all these cases, at any given time, only two of the alleles can occupy the same locus on a pair of homologous chromosomes. Rh factor The Rh factor is an antigen (a protein) that is found on the surface of the red blood cell. Along with the A and B antigens it comprises the major scheme we use for "typing" or classifying human blood for compatibility for transfusion. The term "Rh factor" is short for Rhesus factor. It seems we share this antigen with our relatives in the primate world, the rhesus monkeys. The antigen is either there, or it is not. This is determined by the presence or absence of a single gene. We can test for the presence of the antigen by adding artificially produced antibodies to a mixture of the patient's red cells. If they form clumps, we know that the antibody is present on the red cells. We term this blood Rh positive. If the antigen is not present (and the cells don't clump), we designate the blood type as Rh negative. About the Rh Factor People with different blood types have proteins specific to that blood type on the surfaces of their red blood cells (RBCs). There are four blood types A, B, AB, and O.

Each of the four blood types is additionally classified according to the presence of another protein on the surface of RBCs that indicates the Rh factor. If you carry this protein, you are Rh positive. If you don't carry the protein, you are Rh negative. Most people about 85% are Rh positive. But if a woman who is Rh negative and a man who is Rh positive conceive a baby, there is the potential for a baby to have a health problem. The baby growing inside the Rh-negative mother may have Rh-positive blood, inherited from the father. Approximately half of the children born to an Rh-negative mother and Rh-positive father will be Rh positive. Rh incompatibility usually isn't a problem if it's the mother's first pregnancy because, unless there's some sort of abnormality, the fetus's blood does not normally enter the mother's circulatory system during the course of the pregnancy. However, during delivery, the mother's and baby's blood can intermingle. If this happens, the mother's body recognizes the Rh protein as a foreign substance and can begin producing antibodies (protein molecules in the immune system that recognize, and later work to destroy, foreign substances) against the Rh proteins introduced into her blood. Other ways Rh-negative pregnant women can be exposed to the Rh protein that might cause antibody production include blood transfusions with Rh-positive blood, miscarriage, and ectopic pregnancy. Rh antibodies are harmless until the mother's second or later pregnancies. If she is ever carrying another Rh-positive child, her Rh antibodies will recognize the Rh proteins on the surface of the baby's blood cells as foreign, and pass into the baby's bloodstream and attack those cells. This can lead to swelling and rupture of the baby's RBCs. A baby's blood count can get dangerously low when this condition, known as hemolytic or Rh disease of the newborn, occurs. An antigen present on the red blood cells of about 85% of people. Called Rh because it was first identified in the blood of rhesus monkeys. Persons with the factor are designated Rhpositive, those lacking the factor are designated Rh-negative. Protein on surface of red blood cells in some blood types (Rh+) and absent in others (Rh-). Can cause erythroblastosis fetalis in second pregnancy if fetal/maternal blood of opposite groups mix on first pregnancy. An additional blood factor found in the red blood cells. When it is absent, the person is said to be Rh negative. A group of antigens found on the surface of red blood cells. ----------------------The two most important classifications to describe blood types in humans are ABO and the Rhesus factor (Rh factor). There are 46 other known antigens in humans, most of which are much rarer than ABO and Rh. Blood transfusions from incompatible groups can cause an immunological transfusion reaction, resulting in hemolytic anemia, renal failure, shock, and death. The ABO blood types also exist among chimpanzees and bonobos. The phrases "blood group" and "blood type" are often used interchangeably, although this is not technically correct. "Blood group" is used to refer specifically to a person's ABO status,

while "blood type" refers to both ABO and Rh factors. When typing blood, 'positive' and 'negative' refer to Rh status, not ABO status; thus, for instance, an "A-neg" person is somebody who has the A antigen but does not have the Rh antigen. Blood type is determined by the antigens (epitopes) on the surface of a red blood cell. Some of these are proteins, while others are proteins with polysaccharides attached. The absence of some of these markers leads to production of antibodies against this marker. The exact reason why this happens is poorly understood, as generally an antigen needs to be present to elicit an immune response. Administration of the wrong blood type would lead to immediate destruction of the infused blood. The breakdown products cause acute medical illness; hence it is of vital importance that the blood types of the donor and receptor be properly matched.

Sex-Linked Inheritance In humans, sex is determined by the twenty-third pair of chromosomes known as the sex chromosomes. If at conception you have two X-shaped (XX) chromosomes you are destined to be a female. If you have an X and a Y-shaped chromosome (XY) you are destined to be male. Since the X and the Y chromosomes differ in the information they carry, any gene found on the X chromosome is referred to as a sex-linked gene. Women will have two alleles for sex-linked genes while men will only have one.

Example: Hemophilia is a sex-linked trait. A person with hemophilia is lacking certain proteins that are necessary for normal blood clotting. Since hemophilia is a recessive trait, use N for normal and n for hemophilia. A woman who is heterozygous for hemophilia (a carrier) marries a normal man. What are the possible genotypes of their offspring?

Example: Examine the following pedigree chart of colorblindness. In humans, color blindness is caused by a recessive sex-linked allele. On the diagram, label the genotypes of the individuals.

In humans, there are hundreds of genes located on the X chromosome that have no counterpart on the Y chromosome. The traits governed by these genes thus show sex-linked inheritance. This type of inheritance has certain unique characteristics, which include the following: (1) There is no male-to-male (father-to-son) transmission, since sons will, by definition, inherit the Y rather than the X chromosome. (2) The carrier female (heterozygote) has a 50 percent chance of passing the mutant gene to each of her children; sons who inherit the mutant gene will be hemizygotes and will manifest the trait, while daughters who receive the mutant gene will be unaffected carriers. (3) Males with the trait will pass the gene on to all of their daughters, who will be carriers. (4) Most sex-linked traits are recessively inherited, so that heterozygous females generally do not display the trait. The table lists some sex-linked conditions. The figure shows a pedigree of a family in which a mutant gene for hemophilia A, a sexlinked recessive disease, is segregating. Hemophilia A gained notoriety in early studies of human genetics because it affected at least 10 males among the descendants of Queen Victoria, who was a carrier Hemophilia A, the most widespread form of hemophilia, results from a mutation in the gene encoding clotting factor VIII. Because of this mutation, affected males cannot produce functional factor VIII, so that their blood fails to clot properly, leading to significant and potentially life-threatening loss of blood after even minor injuries. Bleeding into joints commonly occurs as well and may be crippling. Therapy consists of avoiding trauma and of administering injections of purified factor VIII, which was once isolated from outdated human blood donations but can now be made in large amounts through recombinant DNA technology. Although heterozygous female carriers of X-linked recessive mutations generally do not exhibit traits characteristic of the disorder, cases of mild or partial phenotypic expression in female carriers have been reported, resulting from nonrandom X inactivation.

Mutation Mutations are alterations of genetic material. They occur frequently during DNA duplication in cell division. This should not be surprising considering the fact that mitosis and meiosis are essentially mechanical processes with millions of operations that must be precisely completed in order for duplicate DNA molecules to be created. There are four common categories of mutations: 1. DNA base substitutions and deletions 2. unequal crossing-over and related structural modifications of chromosomes 3. partial or complete gene duplication 4. irregular numbers of chromosomes Substitutions and deletions of single bases are common. For example, an adenine can be accidently substituted for a guanine. Such small errors in copying DNA are referred to as point mutations. There is a self correcting mechanism in DNA replication that repairs these small errors, but it does not always find every one of them. Structural modifications of chromosomes generally occur as a consequence of the crossingover process during cell division. Normally, there is an equal exchange of end sections of homologous chromosomes. Occasionally, there is a reunion of an end section onto a chromosome that is not homologous. Likewise, there can be an orphaned end section that does not reattach to any chromosome. The genes on such orphans are functionally lost. Sometimes, extra copies of one or more genes are produced when a DNA molecule is replicated. More often, however, sections of the far more common non-protein coding DNA regions are duplicated. This duplication of large sections of DNA is an important source of genetic variation for a species. Spare copies of genes or inactive genes can mutate and change their function over time thereby producing a new variation that natural selection can favor or reject. Large-scale evolutionary changes in a species line generally occur in this way. Very likely, an explosion of gene duplications 7-12 million years ago led to the branching off of gorillas and then chimpanzees from the evolutionary line that ultimately became modern humans. Irregular numbers of chromosomes can occur as a consequence of errors in meiosis and the combining of parental chromosomes at the time of conception. Such is the case when there are three instead of two autosomes for pair 21. This specific error is characteristic of Down

syndrome.

In order for a mutation to be inherited, it must occur in the genetic material of a sex cell. It is now known that the frequency of new mutations in humans ranges from 100 to 200 for every individual. it is to be expected then that most sex cells also contain gene mutations of some sort. In other words, mutations are probably common occurrences even in healthy people. Most probably do not confer a significant advantage or disadvantage because they are point mutations that occur in non-gene coding regions of DNA molecules. They are relatively neutral in their effect. However, some mutations are extremely serious and can result in death before birth, when an individual is still in the embryonic or early fetal stages of development. Mutations can occur naturally as a result of occasional errors in DNA replication. They also can be caused by exposure to radiation, alcohol, lead, lithium, organic mercury, and some other chemicals. Viruses and other microorganisms may also be responsible for them. Even some commonly prescribed drugs are thought to be mutagens. 1. androgens (steroid hormones that control the development and maintenance of masculine characteristics) 2. ACE inhibitors (a class of blood pressure medication) 3. streptomycin and tetracycline (two classes of antibiotics) 4. vitamin A Mutations appear to be spontaneous in most instances. That does not mean that they occur without cause but, rather, that the specific cause is almost always unknown. Subsequently, it is usually very difficult for lawyers to prove in a court of law that a mutagen is responsible for causing a specific mutation in people. With the aid of expert scientific testimony, they can often demonstrate that the mutagen can cause a particular kind of mutation. However, that is not the same thing as proving that a plaintiff's mutation was caused by that mutagen instead of some others. The great diversity of life forms that have been identified in the fossil record is evidence that there has been an accumulation of mutations producing a more or less constant supply of both small and large variations upon which natural selection has operated for billions of years. Mutation has been the essential prerequisite for the evolution of life. In order for a mutation to be subject to natural selection, it must be expressed in the phenotype of an individual. Selection favors mutations that result in adaptive phenotypes and eliminates nonadaptive ones. Even when mutations produce recessive alleles that are seldom expressed in phenotypes, they become part of a vast reservoir of hidden variability

that can show up in future generations. Such potentially harmful recessive alleles add to the genetic load of a population. Mutation Types of Mutations Knowing a few basic types of mutations can help you understand why some mutations have major effects and some may have no effect at all. Substitution A substitution is a mutation that exchanges one base for another (i.e., a change in a single "chemical letter" such as switching an A to a G). Such a substitution could:

1. change a codon to one that encodes a different amino acid and cause a small change in the protein produced. For example, sickle cell anemia is caused by a substitution in the beta-hemoglobin gene, which alters a single amino acid in the protein produced. 2. change a codon to one that encodes the same amino acid and causes no change in the protein produced. These are called silent mutations. 3. change an amino-acid-coding codon to a single "stop" codon and cause an incomplete protein. This can have serious effects since the incomplete protein probably wont function. Insertion Insertions are mutations in which extra base pairs are inserted into a new place in the DNA.

Deletion Deletions are mutations in which a section of DNA is lost, or deleted.

Frameshift Since protein-coding DNA is divided into codons three bases long, insertions and deletions can alter a gene so that its message is no longer correctly parsed. These changes are called frameshifts. For example, consider the sentence, "The fat cat sat." Each word represents a codon. If we delete the first letter and parse the sentence in the same way, it doesnt make sense. In frameshifts, a similar error occurs at the DNA level, causing the codons to be parsed incorrectly. This usually generates proteins that are as useless as "hef atc ats at" is uninformative.

Genetic drift In population genetics, genetic drift (or more precisely allelic drift) is the statistical effect that results from the influence that chance has on the survival of alleles (variants of a gene). The effect may cause an allele, and the biological trait that it confers, to become more common or rare over successive generations. Ultimately, the drift may either remove the allele from the gene pool or remove all other alleles. Whereas natural selection is the tendency of beneficial alleles to become more common over time (and detrimental ones less common), genetic drift is the fundamental tendency of any allele to vary randomly in frequency over time due to statistical variation alone, so long as it does not comprise all or none of the distribution. Chance affects the commonality or rarity of an allele, because no trait guarantees survival of a given number of offspring. This is because survival depends on non-genetic factors (such as the possibility of being in the wrong place at the wrong time). In other words, even when individuals face the same odds, they will differ in their success. A rare succession of chance events rather than natural selection can thus bring a trait to predominance, causing a population or species to evolve. An important aspect of genetic drift is that its rate is expected to depend strongly on population size as a consequence of the law of large numbers. When many individuals carry a particular allele, and all face equal odds, the number of offspring they collectively produce will only slightly differ from the expected value, which is the expected average per individual times the number of individuals. But with a small effective breeding size, a departure from the norm in one individual causes a disproportionately greater deviation from the expected result. Therefore small populations are subject to more drift than large ones.This is also the basis for the founder effect, a proposed mechanism of speciation. By definition, genetic drift has no preferred direction. A neutral allele may be expected to increase or decrease in any given generation with equal probability. Given sufficiently long time, however, the mathematics of genetic drift (cf. Galton-Watson process) predict the allele will either die out or be present in 100% of the population, after which time there is no random variation in the associated gene. Thus genetic drift tends to sweep gene variants out of a population over time, such that all members of a species would eventually be homozygous for this gene. In this regard, genetic drift opposes genetic mutation which introduces novel variants into the population according to its own random processes. Allele frequencies From the perspective of population genetics, drift is a "sampling effect." To illustrate: on average, coins turn up heads or tails with equal probability. Yet just a few tosses in a row are unlikely to produce heads and tails in equal number. The numbers are no more likely to be exactly equal for many tosses in a row, but the discrepancy in number can be very small (in percentage terms). As an example, ten tosses turn up at least 70% heads about once in every six tries, but the chance of a hundred tosses in a row producing at least 70% heads is only about one in 25,000. Similarly, in a breeding population, if an allele has a frequency of p, probability theory dictates that (if natural selection is not acting) in the following generation, a fraction p of the population will inherit that particular allele. However, as with the coin toss above, allele frequencies in real populations are not probability distributions; rather, they are a random sample, and are thus subject to the same statistical fluctuations (sampling error).

When the alleles of a gene do not differ with regard to fitness, on average the number of carriers in one generation is proportional to the number of carriers in the previous generation. But the average is never tallied, because each generation parents the next one only once. Therefore the frequency of an allele among the offspring often differs from its frequency in the parent generation. In the offspring generation, the allele might therefore have a frequency p', slightly different from p. In this situation, the allele frequencies are said to have drifted. Note that the frequency of the allele in subsequent generations will now be determined by the new frequency p', meaning that drift is a memoryless process and may be modeled as a Markov process. As in the coin toss example above, the size of the breeding population (the effective population size) governs the strength of the drift effect. When the effective population size is small, genetic drift will be stronger. Drifting alleles usually have a finite lifetime. As the frequency of an allele drifts up and down over successive generations, eventually it drifts until fixation - that is, it either reaches a frequency of zero, and disappears from the population, or it reaches a frequency of 100% and becomes the only allele in the population. Subsequent to the latter event, the allele frequency can only change by the introduction of a new allele by a new mutation. The lifetime of an allele is governed by the effective population size. In a very small population, only a few generations might be required for genetic drift to result in fixation. In a large population, it would take many more generations. On average, an allele will be fixed in 4Ne generations, where Ne is the effective population size. According to the Hardy-Weinberg Principle, which holds that allele frequencies in a gene pool will not change over time, a population must be sufficiently large to prevent genetic drift from changing allele frequencies over time. This is why the law is unstable in a small population. Drift versus selection Genetic drift and natural selection rarely occur in isolation from each other; both forces are always at play in a population. However, the degree to which alleles are affected by drift and selection varies according to circumstance. In a large population, where genetic drift occurs very slowly, even weak selection on an allele will push its frequency upwards or downwards (depending on whether the allele is beneficial or harmful). However, if the population is very small, drift will predominate. In this case, weak selective effects may not be seen at all as the small changes in frequency they would produce are overshadowed by drift. Genetic drift in populations Drift can have profound and often bizarre effects on the evolutionary history of a population. These effects may be at odds with the survival of the population. In a population bottleneck, where the population suddenly contracts to a small size (believed to have occurred in the history of human evolution), genetic drift can result in sudden and dramatic changes in allele frequency that occur independently of selection. In such instances, many beneficial adaptations may be eliminated even if population later grows large again. Similarly, migrating populations may see a founder effect, where a few individuals with a

rare allele in the originating generation can produce a population that has allele frequencies that seem to be at odds with natural selection. Founder's effects are sometimes held to be responsible for high frequencies of some genetic diseases.

Epistasis is the interaction between genes. Epistasis takes place when the effects of one gene are modified by one or several other genes, which are sometimes called modifier genes. The gene whose phenotype is expressed is said to be epistatic, while the phenotype altered or suppressed is said to be hypostatic. Epistasis should be distinguished from Dominance, which is an interaction between alleles at the same gene locus. In general, the fitness increment of any one allele depends in a complicated way on many other alleles; but, because of the way that the science of population genetics was developed, evolutionary scientists tend to think of epistasis as the exception to the rule. In the first models of natural selection devised in the early 20th century, each gene was considered to make its own characteristic contribution to fitness, against an average background of other genes. Some introductory college courses still teach population genetics this way. Epistasis and genetic interaction refer to different aspects of the same phenomenon. The term epistasis is widely used in population genetics and refers especially to the statistical properties of the phenomenon, and does not necessarily imply biochemical interaction between gene products. Examples of tightly linked genes having epistatic effects on fitness are found in supergenes and the human major histocompatibility complex genes. The effect can occur directly at the genomic level, where one gene could code for a protein preventing transcription of the other gene. Alternatively, the effect can occur at the phenotypic level. For example, the gene causing albinism would hide the gene controlling color of a person's hair. In another example, a gene coding for a widow's peak would be hidden by a gene causing baldness. Fitness epistasis (where the affected trait is fitness) is one cause of linkage disequilibrium. Studying genetic interactions can reveal gene function, the nature of the mutations, functional redundancy, and protein interactions. Because protein complexes are responsible for most biological functions, genetic interactions are a powerful tool. II Epistasis, the interaction between genes, is a topic of current interest in molecular and quantitative genetics. A large amount of research has been devoted to the detection and investigation of epistatic interactions. However, there has been much confusion in the literature over definitions and interpretations of epistasis. We note that the degree to which statistical tests of epistasis can elucidate underlying biological interactions may be more limited than previously assumed. Epistasis, defined generally as the interaction between different genes, has become a hot topic in complex disease genetics in recent years. For complex traits such as diabetes, asthma, hypertension and multiple sclerosis, the search for susceptibility loci has, to date, been less successful than for simple Mendelian disorders. This is probably due to complicating factors such as an increased number of contributing loci and susceptibility alleles, incomplete penetrance, and contributing environmental effects. The presence of epistasis is a particular cause for concern, since, if the effect of one locus is altered or masked by effects at another locus, power to detect the first locus is likely to be reduced and elucidation of the joint effects at the two loci will be hindered by their interaction. If more than two loci are involved, the situation is likely to be further complicated by the possibility of complex multiway interactions among some or all of the contributing loci.

Epistasis is the best example for gene interaction. It is a pattern of inheritance where a pair of genes situated at one locus, prevent the expression of a pair of genes situated at another locus. Such genes are called inhibiting genes or epistatic genes (Epi = above/over gene, static = standing). It is an intergenic or non allelic form of gene interaction. The basic genes, the expression of which is prevented by the epistatic genes, are called as hypostatic genes. Epistasis reduces the number of phenotypes in the F2 generation of a dihybrid cross. A classical example of epistasis is seen in the white fowls. There are two varieties of white fowls white leg horn and white plymouth rock. A cross between a homozygous, dominant, white leghorn and a homozygous recessive white Plymouth-rock results in a progeny of F1 generation containing dihybrid white progeny and of the F2 generation consists of white and coloured fowls in the ratio of 13:3 in place of the normal phenotypic ratio of 9:3:3:1. In addition, there is a reduction in the number of phenotypes to just two. P1 Phenotype: White Leg Horn Fowl x Plymouth Rock Fowl

CCII WHITE -- - CCIi WHITE-- --CcII WHITE-- ----CcIi WHITE CCIi WHITE ----ccii COLOURED---- CcIi WHITE -- --CciiCOLOURED CcII WHITE-- -- CcIiWHITE --------CcII WHITE-- ----- cCIi WHITE CcIi WHITE ----- Ccii COLOURED -- CcIi WHITE --- cciiWHITE WHITE FOWL: COLOURED FOWL13 : 3 Here, the basic gene C produces colour in the feathers. But the inhibiting gene I prevents the appearance of colour. Gene I interacts with gene C in such a way to suppress its expression. As a result the leg horn fowl is white CCII. The plymouth rock fowl is white since it has recessive genes ccii. In the F1 generation all the progeny are white, but heterozygous (CcIi) when these fowls are allowed to inbreed, in the F2 generation, the genotypes having

gene I along with gene C will produce white fowls. The genotypes which do not have gene I give rise to coloured fowls. Gene Interaction Examples Phenotype------ Phenotypic ratio 1. Coat colour in mouse -- 9:3:4 Black Albino 2. Fruit colour in squash ---12: 3: 1 White: Yellow: Green 3. Flower colour in Pea ---9: 7 Purple: White 4. Fruit shape in squash--- 9:6:1 Disc:Circu!ar:Long 5. Fruit shape in shepherds purse ---15:1 Trianular: Ovoid 6. Feather colour in Fowls---- 3:3 White: Coloured Every individual organism bears several heritable characters. Which are represented by the innumerable genes present on the chromosomes. During meiosis, the chromosomes move into the gametes as units, all the genes present on any given chromosome will segregate as a group and move together from generation to generation. This tendency of the genes located on the same chromosome, to stay together in hereditary transmission, is known as linkage. The genes located on the same chromosome are called linked genes. The principle of linkage was discovered by Bateson and Punnet in 1906 in the sweat pea, plant, Lathyrus odoratus. However, linkage, as a concept was put forth by Thomas Hunt Morgan in 1910 based on his experiment on Drosophila melanogaster. Chromosome Theory of Linkage Morgan, along with Castle formulated the chromosome theory of linkage. It has the following postulates; 1. Genes are found arranged in a linear manner in the chromosomes. 2. Genes which exhibit linkage are located on the same chromosome. 3. Genes generally tend to stay in parental combination, except in cases of crossing over. 4. The distance between linked genes in a chromosome determines the strength of linkage. Genes located close to each other show stronger linkage than that are located far from each other, since the former are less likely to enter into crossing over. Linkage Groups All the genes located on a particular chromosome, form a linkage group. Since the genes present on a particular chromosome have their alleles located on its homologous chromosome, genes on a pair of homologous chromosomes. Hence, the number of linkage groups corresponds to the number of haploid chromosomes found in a species. Drosophila melanogaster has four linkage groups which can be distinguished into three large and one small linkage groups corresponding to the four pairs of chromosomes. Twentythree linkage groups are present in humans corresponding to 23 pairs of chromosomes. Pea plant has seven linkage groups, corresponding to the seven pairs of chromosomes.

Kinds of Linkage Complete Linkage The genes closely located in the chromosome show complete linkage as they have no chance of separating by crossing over and are always transmitted together to the same gamete and the same offspring. Thus, the parental combination of traits is inherited as such by the young one. Incomplete Linkage The genes distantly located in the chromosome show incomplete linkage because they have a chance of separation by crossing over and of going into different gametes and offspring. Genetic linkage occurs when particular genetic loci or alleles for genes are inherited jointly. Genetic loci on the same chromosome are physically close to one another and tend to stay together during meiosis, and are thus genetically linked. This is called autosomal linkage. Alleles for genes on different chromosomes are usually not linked, due to independent assortment of chromosomes during meiosis. Because there is some crossing over of DNA when the chromosomes segregate, alleles on the same chromosome can be separated and go to different daughter cells. There is a greater probability of this happening if the alleles are far apart on the chromosome, as it is more likely that a cross-over will occur between them. The relative distance between two genes can be calculated using the offspring of an organism showing two linked genetic traits, and finding the percentage of the offspring where the two traits do not run together. The higher the percentage of descendants that does not show both traits, the farther apart on the chromosome the two genes are. Among individuals of an experimental population or species, some phenotypes or traits occur randomly with respect to one another in a manner known as independent assortment. Today scientists understand that independent assortment occurs when the genes affecting the phenotypes are found on different chromosomes or separated by a great enough distance on the same chromosome that recombination occurs at least half of the time. An exception to independent assortment develops when genes appear near one another on the same chromosome. When genes occur on the same chromosome, they are usually inherited as a single unit. Genes inherited in this way are said to be linked, and are referred to as "linkage groups." For example, in fruit flies the genes affecting eye color and wing length are inherited together because they appear on the same chromosome. But in many cases, even genes on the same chromosome that are inherited together produce offspring with unexpected allele combinations. This results from a process called crossing over. At the beginning of normal meiosis, a chromosome pair (made up of a

chromosome from the mother and a chromosome from the father) intertwine and exchange sections or fragments of chromosome. The pair then breaks apart to form two chromosomes with a new combination of genes that differs from the combination supplied by the parents. Through this process of recombining genes, organisms can produce offspring with new combinations of maternal and paternal traits that may contribute to or enhance survival. Genetic linkage was first discovered by the British geneticists William Bateson and Reginald Punnett shortly after Mendel's laws were rediscovered. Linkage mapping The observations by Thomas Hunt Morgan that the amount of crossing over between linked genes differs led to the idea that crossover frequency might indicate the distance separating genes on the chromosome. Morgan's student Alfred Sturtevant developed the first genetic map, also called a linkage map. Sturtevant proposed that the greater the distance between linked genes, the greater the chance that non-sister chromatids would cross over in the region between the genes. By working out the number of recombinants it is possible to obtain a measure for the distance between the genes. This distance is called a genetic map unit (m.u.), or a centimorgan and is defined as the distance between genes for which one product of meiosis in 100 is recombinant. A recombinant frequency (RF) of 1 % is equivalent to 1 m.u. A linkage map is created by finding the map distances between a number of traits that are present on the same chromosome, ideally avoiding having significant gaps between traits to avoid the inaccuracies that will occur due to the possibility of multiple recombination events. Linkage mapping is critical for identifying the location of genes that cause genetic diseases. In an ideal population, genetic traits and markers will occur in all possible combinations with the frequencies of combinations determined by the frequencies of the individual genes. For example, if alleles A and a occur with frequency 90% and 10%, and alleles B and b at a different genetic locus occur with frequencies 70% and 30%, the frequency of individuals having the combination AB would be 63%, the product of the frequencies of A and B, regardless of how close together the genes are. However, if a mutation in gene B that causes some disease happened recently in a particular subpopulation, it almost always occurs with a particular allele of gene A if the individual in which the mutation occurred had that variant of gene A and there have not been sufficient generations for recombination to happen between them (presumably due to tight linkage on the genetic map). In this case, called linkage disequilibrium, it is possible to search potential markers in the subpopulation and identify which marker the mutation is close to, thus determining the mutation's location on the map and identifying the gene at which the mutation occurred. Once the gene has been identified, it can be targeted to identify ways to mitigate the disease. Also, the location of a particular gene on a chromosome is called a gene locus. Linkage map A linkage map is a genetic map of a species or experimental population that shows the position of its known genes and/or genetic markers relative to each other in terms of

recombination frequency, rather than as specific physical distance along each chromosome. A genetic map is a map based on the frequencies of recombination between markers during crossover of homologous chromosomes. The greater the frequency of recombination (segregation) between two genetic markers, the farther apart they are assumed to be. Conversely, the lower the frequency of recombination between the markers, the smaller the physical distance between them. Historically, the markers originally used were detectable phenotypes (enzyme production, eye color) derived from coding DNA sequences; eventually, confirmed or assumed noncoding DNA sequences such as microsatellites or those generating restriction fragment length polymorphisms (RFLPs) have been used. Genetic maps help researchers to locate other markers, such as other genes by testing for genetic linkage of the already known markers. A genetic map is not a physical map (such as a radiation reduced hybrid map) or gene map. LOD score method for estimating recombination frequency The LOD score (logarithm (base 10) of odds) is a statistical test often used for linkage analysis in human populations, and also in animal and plant populations. The test was developed by Newton E. Morton. Computerized LOD score analysis is a simple way to analyze complex family pedigrees in order to determine the linkage between Mendelian traits (or between a trait and a marker, or two markers). . Briefly, it works as follows: 1. 2. 3. 4. Establish a pedigree Make a number of estimates of recombination frequency Calculate a LOD score for each estimate The estimate with the highest LOD score will be considered the best estimate

The LOD score is calculated as follows:

NR denotes the number of non-recombinant offspring, and R denotes the number of recombinant offspring. The reason 0.5 is used in the denominator is that any alleles that are completely unlinked (e.g. alleles on separate chromosomes) have a 50% chance of recombination, due to independent assortment. Theta is the recombinant fraction, it is equal to R / (NR + R) In practice, LOD scores are looked up in a table which lists LOD scores for various standard

pedigrees and various values of recombination frequency. By convention, a LOD score greater than 3.0 is considered evidence for linkage. (A score of 3.0 means the Likelihood of observing the given pedigree if the two loci are not linked is less than 1 in 1000). On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage. Although it is very unlikely that a LOD score of 3 would be obtained from a single pedigree, the mathematical properties of the test allow data from a number of pedigrees to be combined by summing the LOD scores. Recombination frequency Recombination frequency () is the frequency that a chromosomal crossover will take place between two loci (or genes) during meiosis. Recombination frequency is a measure of genetic linkage and is used in the creation of a genetic linkage map. A centimorgan (cM) is a unit that describes a recombination frequency of 1%. During meiosis, chromosomes assort randomly into gametes, such that the segregation of alleles of one gene is independent of alleles of another gene. This is stated in Mendel's Second Law and is known as the law of independent assortment. The law of independent assortment always holds true for genes that are located on different chromosomes, but for genes that are on the same chromosome, it does not always hold true. As an example of independent assortment, consider the crossing of the pure-bred homozygote parental strain with genotype AABB with a different pure-bred strain with genotype aabb. A and a and B and b represent the alleles of genes A and B. Crossing these homozygous parental strains will result in F1 generation offspring with genotype AaBb. The F1 offspring AaBb produces gametes that are AB, Ab, aB, and ab with equal frequencies (25%) because the alleles of gene A assort independently of the alleles for gene B during meiosis. Note that 2 of the 4 gametes (50 %)Ab and aBwere not present in the parental generation. These gametes represent recombinant gametes. Recombinant gametes are those gametes that differ from both of the haploid gametes that made up the diploid cell. In this example, the recombination frequency is 50% since 2 of the 4 gametes were recombinant gametes. The recombination frequency will be 50% when two genes are located on different chromosomes or when they are widely separated on the same chromosome. This is a consequence of independent assortment. When two genes are close together on the same chromosome, they do not assort independently and are said to be linked. Whereas genes located on different chromosomes assort independently and have a recombination frequency of 50%, linked genes have a recombination frequency that is less than 50%. As an example of linkage, consider the classic experiment by William Bateson and Reginald Punnett. They were interested in trait inheritance in the sweet pea and were studying two genesthe gene for flower color (P, purple, and p, red) and the gene affecting the shape of

pollen grains (L, long, and l, round). They crossed the pure lines PPLL and ppll and then self-crossed the resulting PpLl lines. According to Mendelian genetics, the expected phenotypes would occur in a 9:3:3:1 ratio of PL:Pl:pL:pl. To their surprise, they observed an increased frequency of PL and pl and a decreased frequency of Pl and pL (see table below). Bateson and Punnett experiment...... Phenotype and genotype....... Observed Expected from 9:3:3:1 ratio Purple, long (PpLl)..... 284..... 216 Purple, round (Ppll)..... 21..... 72 Red, long (ppLl).......... 21..... 72 Red, round (ppll) ......... 55 .....24 Their experiment revealed linkage between the P and L alleles and the p and l alleles. The frequency of P occurring together with L and with p occurring together with l is greater than that of the recombinant Pl and pL. The recombination frequency cannot be computed directly from this experiment, but intuitively it is less than 50%. The progeny in this case received two dominant alleles linked on one chromosome (referred to as coupling or cis arrangement). However, after crossover, some progeny could have received one parental chromosome with a dominant allele for one trait (eg Purple) linked to a recessive allele for a second trait (eg round) with the opposite being true for the other parental chromosome (eg red and Long). This is referred to as repulsion or a trans arrangement. The phenotype here would still be purple and long but a test cross of this individual with the recessive parent would produce progeny with much greater proportion of the two crossover phenotypes. While such a problem may not seem likely from this example, unfavorable repulsion linkages do appear when breeding for disease resistance in some crops. When two genes are located on the same chromosome, the chance of a crossover producing recombination between the genes is related to the distance between the two genes. Thus, the use of recombination frequencies has been used to develop linkage maps or genetic maps.

Genetic Engineering Genetic engineering is a laboratory technique used by scientists to change the DNA of living organisms. DNA is the blueprint for the individuality of an organism. The organism relies upon the information stored in its DNA for the management of every biochemical process. The life, growth and unique features of the organism depend on its DNA. The segments of DNA which have been associated with specific features or functions of an organism are called genes. Molecular biologists have discovered many enzymes which change the structure of DNA in living organisms. Some of these enzymes can cut and join strands of DNA. Using such

enzymes, scientists learned to cut specific genes from DNA and to build customized DNA using these genes. They also learned about vectors, strands of DNA such as viruses, which can infect a cell and insert themselves into its DNA. With this knowledge, scientists started to build vectors which incorporated genes of their choosing and used the new vectors to insert these genes into the DNA of living organisms. Genetic engineers believe they can improve the foods we eat by doing this. For example, tomatoes are sensitive to frost. This shortens their growing season. Fish, on the other hand, survive in very cold water. Scientists identified a particular gene which enables a flounder to resist cold and used the technology of genetic engineering to insert this 'anti-freeze' gene into a tomato. This makes it possible to extend the growing season of the tomato. enetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism's genes. Genetic engineering is different from traditional breeding, where the organism's genes are manipulated indirectly. Genetic engineering uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. Genetic engineering techniques have found some successes in numerous applications. Some examples are in improving crop technology, the manufacture of synthetic human insulin through the use of modified bacteria, the manufacture of erythropoietin in hamster ovary cells, and the production of new types of experimental mice such as the oncomouse (cancer mouse) for research. The term "genetic engineering" was coined in Jack Williamson's science fiction novel Dragon's Island, published in 1951, two years before James Watson and Francis Crick showed that DNA could be the medium of transmission of genetic information. There are a number of ways through which genetic engineering is accomplished. Essentially, the process has five main steps. 1. Isolation of the genes of interest 2. Insertion of the genes into a transfer vector 3. Transfer of the vector to the organism to be modified 4. Transformation of the cells of the organism 5. Selection of the genetically modified organism (GMO) from those that have not been successfully modified Isolation is achieved by identifying the gene of interest that the scientist wishes to insert into the organism, usually using existing knowledge of the various functions of genes. DNA information can be obtained from cDNA or gDNA libraries, and amplified using PCR techniques. If necessary, i.e. for insertion of eukaryotic genomic DNA into prokaryotes, further modification may be carried out such as removal of introns or ligating prokaryotic promoters. Insertion of a gene into a vector such as a plasmid can be done once the gene of interest is isolated. Other vectors can also be used, such as viral vectors, bacterial conjugation, liposomes, or even direct insertion using a gene gun. Restriction enzymes and ligases are of great use in this crucial step if it is being inserted into prokaryotic or viral vectors. Daniel Nathans, Werner Arber and Hamilton Smith received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases. Once the vector is obtained, it can be used to transform the target organism. Depending on the vector used, it can be complex or simple. For example, using raw DNA with gene guns is

a fairly straightforward process but with low success rates, where the DNA is coated with molecules such as gold and fired directly into a cell. Other more complex methods, such as bacterial transformation or using viruses as vectors have higher success rates. After transformation, the GMO can be selected from those that have failed to take up the vector in various ways. One method is screening with DNA probes that can stick to the gene of interest that was supposed to have been transplanted. Another is to package genes conferring resistance to certain chemicals such as antibiotics or herbicides into the vector. This chemical is then applied ensuring that only those cells that have taken up the vector will survive. Applications The first genetically engineered medicine was synthetic human insulin, approved by the United States Food and Drug Administration in 1982. Another early application of genetic engineering was to create human growth hormone as replacement for a compound that was previously extracted from human cadavers. In 1987 the FDA approved the first genetically engineered vaccine for humans, for hepatitis B. Since these early uses of the technology in medicine, the use of GM has gradually expanded to supply a number of other drugs and vaccines. One of the best-known applications of genetic engineering is the creation of GMOs for food use (genetically modified foods); such foods resist insect pests, bacterial or fungal infection, resist herbicides to improve yield, have longer freshness than otherwise, or have superior nutritional value. In materials science, a genetically modified virus has been used to construct a more environmentally friendly lithium-ion battery. A new type of slowly growing artform is being established via gene engineering and manipulation. Bioart, an artistic form, uses gene engineering to create new art forms that both educate the public about genetics and create living artforms. During the latter stage stages of the 20th century, man harnessed the power of the atom, and not long after, soon realised the power of genes. Genetic engineering is going to become a very mainstream part of our lives sooner or later, because there are so many possibilities advantages (and disadvantages) involved. Here are just some of the advantages : * Disease could be prevented by detecting people/plants/animals that are genetically prone to certain hereditary diseases, and preparing for the inevitable. Also, infectious diseases can be treated by implanting genes that code for antiviral proteins specific to each antigen. * Animals and plants can be 'tailor made' to show desirable characteristics. Genes could also be manipulated in trees for example, to absorb more CO2 and reduce the threat of global warming. * Genetic Engineering could increase genetic diversity, and produce more variant alleles which could also be crossed over and implanted into other species. It is possible to alter the genetics of wheat plants to grow insulin for example.

Of course there are two sides to the coin, here are some possible eventualities

and disadvantages. * Nature is an extremely complex inter-related chain consisting of many species linked in the food chain. Some scientists believe that introducing genetically modified genes may have an irreversible effect with consequences yet unknown. * Genetic engineering borderlines on many moral issues, particularly involving religion, which questions whether man has the right to manipulate the laws and course of nature. Genetic engineering may be one of the greatest breakthroughs in recent history alongside the discovery of the atom and space flight, however, with the above eventualities and facts above in hand, governments have produced legislation to control what sort of experiments are done involving genetic engineering. In the UK there are strict laws prohibiting any experiments involving the cloning of humans. However, over the years here are some of the experimental 'breakthroughs' made possible by genetic engineering. * At the Roslin Institute in Scotland, scientists successfully cloned an exact copy of a sheep, named 'Dolly'. This was the first successful cloning of an animal, and most likely the first occurrence of two organisms being genetically identical. Note : Recently the sheep's health has deteriorated detrimentally * Scientists successfully manipulated the genetic sequence of a rat to grow a human ear on its back. (Unusual, but for the purpose of reproducing human organs for medical purposes) * Most controversially, and maybe due to more liberal laws, an American scientist is currently conducting tests to clone himself. Genetic engineering has been impossible until recent times due to the complex and microscopic nature of DNA and its component nucleotides. Through progressive studies, more and more in this area is being made possible, with the above examples only showing some of the potential that genetic engineering shows. For us to understand chromosomes and DNA more clearly, they can be mapped for future reference. More simplistic organisms such as fruit fly (Drosophila) have been chromosome mapped due to their simplistic nature meaning they will require less genes to operate. At present, a task named the Human Genome Project is mapping the human genome, and should be completed in the next ten years. The process of genetic engineering involves splicing an area of a chromosome, a gene, that controls a certain characteristic of the body. The enzyme endonuclease is used to split a DNA sequence and split the gene from the rest of the chromosome. For example, this gene may be programmed to produce an antiviral protein. This gene is removed and can be placed into another organism. For example, it can be placed into a bacteria, where it is sealed into the DNA chain using ligase. When the chromosome is once again sealed, the bacteria is now effectively re-programmed to replicate this new antiviral protein. The bacteria can continue to live a healthy life, though genetic engineering and human intervention has actively manipulated what the bacteria actually is. No doubt there are advantages and disadvantages, and this whole subject area will become more prominent over time.

Genetic engineering, more formally known as recombinant DNA technology, allows scientists to pluck genes (segments of DNA) from one type of organism and combine them with genes

of a second organism. In this way, relatively simple organisms such as bacteria or yeast, or even mammalian cells in culture and mammals such as goats and sheep, can be induced to make quantities of human proteins, including hormones such as insulin as well as lymphokines and monokines. Microorganisms can also be made to manufacture proteins from infectious agents such as the hepatitis virus or the AIDS virus, for use in vaccines. Another facet of recombinant DNA technology involves gene therapy: replacing defective or missing genes with normal genes. The first approved gene therapy trials involved children with severe combined immunodeficiency disease, or SCID (Immunodeficiency Diseases), which is caused by lack of an enzyme due to a single abnormal gene. The missing gene is introduced into a harmless virus, then mixed with progenitor cells from the patient's bone marrow. When the virus splices its genes into those of the bone marrow cells, it simultaneously inserts the gene for the missing enzyme. Injected back into the patient, the treated marrow cells produce the missing enzyme and revitalize the immune defenses. Researchers are also investigating the use of gene therapy for such diverse conditions as hemophilia, Parkinson's disease, diabetes, a hereditary form of dangerously high cholesterol, and AIDS. An increasingly important target for gene therapy is cancer. In pioneering experiments, scientists are removing the immune cell known as the tumor-infiltrating lymphocyte or TIL(Immunity and Cancer), or tumor cells themselves, inserting a gene that boosts the cells' ability to make quantities of a natural anticancer product such as tumor necrosis factor (TNF) or interleukin-2, and then growing the restructured cells in quantity in the laboratory. When the altered cells are returned to the patient, they seek out the tumor and deliver large doses of the anticancer chemical. They also appear to mobilize, in some unknown way, additional antitumor defenses. On the horizon are anticancer vaccines made by manipulating genes. Intended to protect cancer patients against a recurrence, these vaccines can incorporate genes for immunogenic tumor antigens or genes for histocompatibility antigens able to galvanize killer T cells, as well as genes for substances such as TNF or interleukin-2. Other anticancer strategies call for introducing genes that can shut down cancer-promoting oncogenes or replace faulty cancer-restraining suppressor genes. Genes can be packaged, for delivery, in a variety of ways: inserted into the genetic material of such carriers as the familiar vaccinia virus (Vaccines Through Biotechnology) or inactivated retroviruses, grafted onto a protein carrier that magnifies the immune response (an adjuvant), or tucked into fat globules known as liposomes.

Introduction: What is Genetic Engineering? Who would have thought that a tomato could possess characteristics of a fish? What about a plant possessing characteristics of a firefly; or a pig with human traits? These things may sound like science experiments gone wrong, but in truth, these are products of experiments that went well. The fish-like tomato and others are results of genetic modification or genetic manipulation, which are more commonly known as genetic engineering. Genetic engineering is the process of taking genes and segments of DNA from one species and putting them into another species, thus breaking the species barrier and artificially modifying the DNA of various species. These changes in DNA result in an alteration of reproductive and hereditary processes of the organisms since the process is irreversible and the organism's offspring will also possess this unique DNA .

The Process of Genetic Engineering

In order to understand how genetic manipulation is accomplished, it is important first to understand the structure of deoxyribonucleic acid, or DNA. Within its chemical structure, DNA stores the information that determines an organism's hereditary or genetic properties. DNA is made up of a linked series of units called nucleotides , Different nucleotide sequences determine different genes genetic information. Genetic engineering is based on this genetic information. Genetic manipulation is carried out through a process known as recombinant-DNA formation, or gene splicing. This procedure behind genetic engineering is one whereby segments of genetic material from one organism are transferred to another. The basis of the technique lies in the use of restriction enzymes that split DNA strands wherever certain desired secjuences of nucleotides, or specific genes, occur. This desired segment of DNA is referred to as donor DNA. The process of gene splicing results in a series of fragments of DNA, each of which express the same desired gene that can then combine with plasmids ). Plasmids are small, circular molecules of DNA that are found in many bacteria. The bacteria act as vectors in the process of genetic engineering. The desired gene cannot be directly inserted into the recipient organism, or host, therefore there must be an organism that can carry the donor DNA into the host. Plasmid DNA is isolated from bacteria and its circular structure is broken by restriction enzymes (Dworkin). The desired donor DNA is then inserted in the plasmid, and the circle is resealed by ligases, which are enzymes that repair breaks in DNA strands. This reconstructed plasmid, which contains an extra gene, can be replaced in the bacteria, where it is cloned, or duplicated, in large numbers. The combined vector and donor DNA fragment constitute the recombinant-DNA molecule. Once inside a host cell, this molecule is replicated along with the host's DNA during cell division. These divisions produce a clone of identical cells, each having a copy of the recombinant-DNA molecule and thus permanently changing the genetic makeup of the host organism ). Genetic engineering has been accomplished. The Many Uses of Genetic Engineering Are there any benefits that genetic engineering could bring to humankind? Actually, there are many. By performing genetic engineering, scientists can obtain knowledge about genetic mechanisms. For example, they may be able to uncover some secrets of genetic mapping. Genetic mapping is the identification of individual genes for various functions. If scientists are rising restriction enzymes to splice certain genes, they must be able to identify the genes. Thus, genetic engineering helps to identify certain nucleotide sequences, and to use various restriction enzymes to "read" the sequences. For example, if it appears that a single gene is responsible for a certain function, the recombinant-DNA process may tell us otherwise that two multiple genes, or even other factors are responsible for the specific function . Genetic manipulation is most commonly used to transfer desirable qualities from one organism to another to improve the ability of other species to serve humankind. Many examples of this lie in the use of genetic engineering to solve many problems with regards to food production and agriculture, waste disposal and industry, as well as disease and

medicine . The processes are also used for examining evolutionary processes. Food Production and Agriculture Dozens of food crops have now been genetically altered to enhance their qualities for the market or improve growing characteristics. Recombinant-DNA has been used to combat one of the greatest problems in plant food production: the destruction of crops by plant viruses as well as the weather. By transferring the protein-coat gene of the zucchini yellow mosaic virus to squash plants that had previously sustained great damage from the virus, scientists were able to create transgenic squash plants with immunity to this virus . Scientists have also developed transgenic bacteria that protect strawberry plants from injury by frost. The bacteria commonly found on strawberry plants secrete a protein that initiates the formation of ice crystals when the temperature falls to freezing . In the genetically modified bacteria, the gene that codes for the protein has been deleted. In the absence of the protein, ice formation does not occur until the temperature falls well below the freezing point. Normally, such a deep drop in the temperature does not occur until after the harvest period has ended. The first field test of these genetically modified bacteria was conducted in 1987, on a plot of strawberry plants, and similar experiments on potatoes showed that the gene-spliced bacteria were effective in establishing themselves on the plants and, later in the season, in preventing ice formation during periods of light frost (Levine). Genetic engineering has been used in plants as well as in animals. In the livestock industry, for example, large amounts of a growth hormone found in cows have been obtained from genetically engineered bacteria. When treated with this hormone, dairy cows produce more milk, and beef cattle have leaner meat. Similarly, a genetically engineered pig hormone causes hogs to grow faster and decreases fat content in pork).

Waste Disposal and Industry Gene transfers also have been applied in the management of industrial wastes. Genetically altered bacteria can be used to decompose many forms of garbage and to break down petroleum products. For example, an 'oil-eating "nonnatural manmade microorganism" exists, and is used for cleaning up oil spills. Recombinant-DNA technology also can be used to monitor the breakdown of pollutants. For example, naphthalene, an environmental pollutant present in artificially manufactured soils, can be broken down by the bacterium Pseudomonas fluorescens. To monitor this process, scientists transferred a lightproducing enzyme called luciferase, found in the bacterium Vibrio fischeri, to the Pseudornonas fluorescens bacterium. The genetically altered Pseudomonas fluorescens bacterium produces light in proportion to the amount of its activity in breaking down the naphthalene, thus providing a way to monitor the efficiency of the process .

Disease and Medicine Genetic engineering has been used in the field of medicine for many purposes regarding the

control and improvement of health. The process has been used to correct inherited genetic defects causing disease (gene therapy), to counter effects of genetic mutations, to produce various pharniaceutical products . Gene therapy is the use of genetic engineering techniques in the treatment of a genetic disorder or chronic disease. In 1990, a four-year-old girl received genie therapy treatment for adenosine deaminase (ADA) deficiency, an ordinarily fatal inherited disease of the immune system. Because of this genetic defect, the girl was susceptible to recurrent lifethreatening infections. Doctors removed white blood cells from the child's body, let the cells grow in the lab, used a genetically modified virus to carry a normal ADA gene into her inimune cells, and then infused the genetically modified blood cells back into the patient's bloodstream. The inserted ADA gene then programmed the cells to produce the missing ADA enzyme, which led to normal immune function iii those cells. This treatment temporarily helped her to develop resistance to infection, and must be repeated periodically . Another important medical application of the recombinant- DNA procedure has been the production of vaccines against a number of diseases. Heretofore, vaccination against a disease has involved the injection of killed or weakened microorganisms into a person, with the subsequent production of antibodies by the individual's immune system. This procedure has always carried the risk of there being live, virulent pathogens in the vaccine because of some error in the vaccine-producing process (Donnelly). Through the recombinant-DNA procedure, it is now possible to transfer the genes that stimulate antibody formation to a harmless microorganism and use it as a vaccine against the particular disease. Vaccines have been successfully created using the harmless cowpox virus, the herpes simplex type I virus (cold sores), the influenza virus, and the hepatitis B virus through gene splicing . Genetic engineering has also contributed several pharmaceutical products (besides vaccinations). Recombinant-DNA procedures involving bacteria and donor DNA fragments have led to the increased availability of such medically important substances as insulin, interferon, and growth hormone (Rubenstein). These substances were previously available only in limited quantities from their primary sources. Insulin is a hormone produced in the pancreas that controls the absorption of glucose by cells. Diabetics lack the hormone or have decreased levels of it. Using recombinant-DNA techniques, scientists have created human insulin, which is artificially produced by genesplicing methods in bacteria. Heretofore, diabetic patients relied solely on insulin derived from the pancreases of animals to control glucose levels . The protein interferon is released into the bloodstream to induce healthy cells to manufacture an enzyme that counters a viral infection. It can also be effective against some forms of cancer, leukemia, genital warts, and the common cold. For many years, the supply of human interferon for research was limited by costly extraction techniques. l-Iowever, the protein became available in greater quantities through genetic engineering .

Evolution Surprisingly, genetic engineering can also be used to uncover the past. Recombinant-DNA procedures have been used to study the genes of extinct animals. A zebra-like animal called the quagga, for example, became extinct in the '1 9~ century, but some quagga skins with underlying muscle tissues have been salt-preserved in museums. Enzymes were used to release DNA from these muscle cells, yielding DNA fragments representing parts of different genes. These fragments were transferred to the plasmids of bacteria, where they were replicated along with the bacterial DNA. They were then retrieved, analyzed, and compared to corresponding DNA segments of closely related living animals, revealing that the quagga DNA differs from its zebra counterpart by about 5 percent. This amount of difference indicates that the quagga and zebra shared a common ancestor. With appropriate modifications, it should be possible to use this technique to study genes from the bones and teeth of long-extinct animals, providing new insights into the evolutionary process .

The Dangers of Genetic Engineering Even though it may seem as if genetic engineering has so many positive aspects, there are just as many negatives to counter. Genetic engineering is really a test tube science which may be prematurely applied. A gene studied under a closed system, a test tube with no outside influence on the conditions of the experiment, can only give results about what it does and how it behaves in that particular system. The experiment cannot tell what the role and behavior of donor DNA will be once it's in the host cell, which is likely to be a totally unrelated species that may be very different from the experimental environment . The insertion of a foreign gene might trigger new cellular activities or interrupt current ones. For example, genes can normally be exchanged between different species, but the frequency of these natural transfers is limited by their defense (immune) systems. The immune system serves to prevent invasion by harmful foreign genes, viruses, and other substances, so that particular species is able to maintain its characteristic traits and norma] metabolism . Genetic engineering, may, in turn, disrupt and weaken the immune systems by introducing foreign substances into organisms that they won't be able to fight. No one really knows the overall effect of this . Foreign genes trigger new cellular activities in the form of resistance. The vectors used in the genetic engineering process are often resistant to many drugs such as antibiotics. Injecting a drug-resisting vector into a new organism will result in a drug-resistant host organism. The resistance may not necessarily be only to drugs, as is the case with frostresistant plants. Since genetic engineering is irreversible, this method allows these altered organisms to become widespread in nature. Creating organisms that are resistant to anything they weren't initially unaffected by disturbs the evolutionary process of natural selection, or survival of the fittest. Putting such a desirable gene into an organism may give them an edge over another that would not

normally exist. This whole idea of meddling with nature raises a question of religion and ethics (Rubenstein). What about "God;" Do we have the right to be playing the role of a higher being? In fact, genetic engineering raises countless more unanswered questions. Should animals be used in research? Do animals have "rights", as we think of "human rights?" How does genetic engineering of food affect religious and other groups with strong dietary laws, such as vegetarians? How great are the potential risks involved in releasing genetically modified organisms into the biosphere without knowing all the possible consequences? Other dangers of genetic engineering include the following: * New toxins and allergens in foods as well as other damaging effects on health caused by unnatural foods: The process of genetic engineering can thus introduce dangerous new allergens and toxins into foods that were previously naturally safe. Already, one genetically engineered soybean was found to cause serious allergic reactions, and bacteria genetically engineered to produce large amounts of the food supplement tryptophan have produced toxic contaminants that have killed 37 people and permanently disabled 1,500 more . * The disturbance of ecological balance and the spread of diseases across species barriers: When genetic engineers insert a new gene into any organism, there are "position effects" which can lead to unpredictable changes in the pattern of genetic function. The protein product of the inserted gene may carry out unexpected reactions and produce potentially toxic products. There is also serious concern about the dangers of using genetically engineered viruses as vectors in the generation of transgenic plants and animals. This could destabilize the genome, and also possibly create new viruses, and thus dangerous new diseases . * Unnatural loss of bio-diversity in crops: Biotechnology companies claim that their manipulations are similar to natural genetic changes or traditional breeding techniques. However, the cross-species transfers being made, such as between fish and tomatoes, or between other unrelated species, would not happen in nature and may create new toxins, diseases and weaknesses (Rubenstein). Also consider the fact that organisms are "sharing" characteristics that are supposed to be unique to them. , For example in the fish-like tomato, a fish and a tomato are no longer unique. There is less diversity since they are now more similar, though unnaturally, than they initially were. * The creation of herbicide-resistant weeds, resulting in increased pollution of food and water supply: More than 50% of the crops developed by biotechnology companies have been engineered to be resistant to herbicides. Use of herbicide-resistant crops will lead to a threefold increase in the use of herbicides, resulting in even greater pollution of our food and water with toxins. * Unexpected characteristics may appear in genetically altered organisms:One batch of genetically engineered young salmon were pale green instead of the normal brown color of young salmon and rather than turning pink on maturation, they remained green. * Artificially induced characteristics and inevitable side effects will be passed on to all subsequent generations and to other related organisms. Once released, they can never be recalled or contained. The consequences of this are incalculable ). Even when genetically engineered fish have appeared normal, their descendants have been born with deformities such as grotesquely deformed heads and gill flaps, and change of color .

Regulation of Genetic Engineering Although numerous dangers of genetic engineering exist, they have been highly exaggerated. There is a great need to understand the genetic basis of all diseases, not just those to be errors of experiments. For genetic diseases, the solution lies in curing the defects rather than in managing with lifelong and often inadequate treatments. Vaccines have been developed through genetic engineering and have virtually eliminated some of mans most dreaded diseases. Even though vaccines do kill or maim a small number of those inoculated, the exaggeration of the downsides and possible dangers in these experiments diverts attention from the research needed to solve these problems and prevent others . The potential dangers of such research must be balanced against the actual tragedies caused by other sources. In an effort to prevent worldwide epidemics and other problems as a result of genetic engineering, the National Institutes of Health (NIH) has established regulations, and has published safety guidelines to minimize the hazards of research . These guidelines have been gradually relaxed because such research was proven to be safe. In 1985, the NIH approved experimental guidelines for treatment in which genes are transplanted to correct hereditary defects in human beings (gene therapy). In 1987, a committee of the National Academy of Sciences concluded that transferring genes between species of organisms posed no serious environmental hazards ).

Conclusion Genetic engineering is a complicated process that can be used for many things from modifying organisms such as plants to serve humankind better, to developing helpful pharmaceutical products, and even providing clues to the evolutionary process . Despite the fact that the genetic manipulation process seems to result in more damage than help, these views are often exaggerated. Although no one can predict the future of any field of human endeavor, genetic engineering appears to be a feasible mechanism through which many problems of modern society can be solved.