Modern Histological Research

Introduction- IVTC as a Technique
It has taken decades to develop the in vivo cryotechnique (IVCT), which is currently being used for examination of the functioning of morphology in the living animals and other organisms. Using IVTC the animals and organisms are cryofixed while the blood is still circulating, this way all the soluble components in the body fluids and structural features of the organism in vivo are easily captured in the frozen tissue specimen. The morphological data from the experiment represents the living state of the organisms cells when it was cryofixed. It has emerged in the recent years that IVCT could be used for immunohistochemical studies and also in the analysis of translocated signal molecules. IVCT has not been used for analysis of liver in living organisms. But in the present study the ever changing liver of a mouse is examined using different hemodynamic conditions of IVCT. The results from this experiment were compared with the one from the conventional chemical-fixation and QF In Vivo Cryotechnique (IVCT) to View Mouse Liver.

Using In Vivo Cryotechnique (IVCT) to examine the mouse liver with ultrastructural, immunohistochemical and histochemical analyses sinusoids are widely opened with numerous erythrocytes flowing in samples prepared by IVCT, this was observed under normal circulation. It was also observed that they were collapsing in arrested mice. On the other hand the sinusoidal cavities were dilated by perfusion-fixation and were observed to have collapsed by immersion-fixation and quick-freezing (QF) methods. The immunoreactivity of the serum albumin and immunoglobulin G and intensity of periodic acid-Schiff-straining in hepatocytes were well preserved with the QF method and IVCT. Then the tissue resection was rapidly converted into hepatocytes as was demonstrated by immunoreactions on the QF tissue frozen 1or 5mm after resection. In the IVCT sample the translocation was not observed this is an indication that IVCT might be important in the examination of cell membrane permeability of hepatocytes in different pathological environments. Immunodistribution and dynamic morphology of the soluble parts in the living organisms (mouse liver) showing the pathological and the physiological states can be precisely examined by in vivo cryotechnique (IVCT) producing higher resolution/

Purpose of Experiment
Both immunohistochemical and Morphological in the analyses of the livers of organisms and animals under abnormal blood circulation and normal conditions would be necessary as they yield important results that reflect the physiology andor pathology of the human liver.

Technique and Procedure
Perfusion-fixation and the alcohol-dehydration (PF-DH) After four mice had been starved for one day, then they were anaesthetized with inhalation of diethyl ether. Then liver was resected and immersed in the same fixative for a night embedded in paraffin wax.

Immersion- fixation process followed by alcohol-dehydration (IM-DH) After the mice were removed from the embedment of the paraffin they were buffered with PFA for microscopy. The specimens were embedded in paraffin wax for magnification to be achieved and those that were to be viewed under electron microscope were embedded in epoxy resins.

Quick- freezing process resected tissues that were freshly obtained (FQF) After the above procedure was achieved, the left lobe of the four specimen were removed from the four specimen under anesthesia and were then chipped into small pieces which were immersed into isopentane for freezing to take place. This process was followed by imbedding the pieces in paraffin wax.

IVCT under varying conditions of hemodynamic In this step sixteen specimens were required and thus 16 mice were anaesthetized and their left lobes were exposed as previously described above. The first four mice livers were cryofixed with IVCT. Four other mice were used in another experiment before IVCT their portal veins and hepatic arteries were ligated at 80min (IVCT-IC), then sham control was performed on another four mice and lastly the four final group of mice were sacrificed by introduction of excessive presence of anesthesia before IVCT, heart arresting.

Preparation of the tissues by PF-DH, IM-DH, FQF and perfusion-diffusion followed by quick freezing (PF-QF) During this step four mice that had not been starved were anaesthetized, their four left lobes were resected.

Conclusions Based on the Experiment Findings
In conclusion, IVCT is used in examination of dead morphology but the dynamic and functional living morphology of mouse livers under various physiological or pathological conditions. It is thus necessary to preserve soluble components like proteins and glycogen in hepacytes which were lost through routine chemical-fixation methods and was also used in the analysis of changes of permeability in cell membranes in hepacytes (Ohao, Terada and Ohno 384).

Morphological Analysis of Sinusoids
In the experiment above the sinusoidal cavities were found to be opened and they contained flowing erythrocytes. For FQF 1min the erythrocytes were almost fully collapsed. It was more obvious here than it was in IVCT-IC and after the sham no results were reflected. With congested erythrocytes they were found to be wider when under heart arrest. PF-DH, there were no erythrocytes observed but the sinusoids were widely opened.

Immunohistochemistry Results
One important benefit for IVCT freezing for organs in living organism is that tissue resection can be achieved without stoppage of blood circulation. The functioning structures of the living tissues that change under varying hemodynamic conditions can be detected by IVCr.

Results of PAS Staining
In the first experiment PAS products from all lobular were preserved by FQF but were partially lost in the peripheral cytoplasm around the centrilobular sinusoids PF-QF. There was more glucose in the periportal areas (PF-DF) and the PAS staining in the cytoplasm was weak in the hepatocytes that are located in the mid centrilobular areas (Ohao, Terada and Ohno 386).

The specimens for PAS staining are prepared through varying methods. Examination of soluble glycogen in hepatecytes by PAS staining of specimens that were prepared by IVCT, FQF and PF-QF followed. To minimize the differences in glycogen content in the tissue blocks, the liver specimens were taken from the same lobe of the mouse and they were examined under the same conditions.

Conclusions Drawn from Results of Experiment
It can be concluded that, IVCT enables us to examine the functionality and dynamicity of the living morphology of the mouse livers under different physiological and pathological conditions and is also important in preservation of proteins and glycogen in hepatocytes.

Extracellular Space in Mouse Cerebellar Cortex under In Vivo Cryotechnique
The method for preparing tissue specimen to be used for morphological analysis of the central nervous the convectional method for preparing tissue specimen for the morphological analysis of the central nervous system. The current study is done by analysis of ultra structures of the cerebellar cortex of the mouse. Also, Purkinje cell layers and molecular structures of the mouse cerebellum are prepared among cellular profiles and synaptic clefts.

Purpose of Experiment
The purpose of features of IVCr for the morphological analysis in vivo is the essence to obtain the analysis from the experiment on the mouse cerebellar cortex with anoxia caused by few artifacts during tissue preparation steps by the use of IVCr.

Conclusions Based on Experiment Findings
There are not much differences in the mean perisynaptic ECS between opened and enclosed synapses. The difference that suggests that the glial processes ensheathing synapses might extend the entire open perisynaptic ECS with a few structural changes to the nearby structures area insignificant. The glial cells observed to affect synaptic neurotransmission, also related to the structural development and restructuring of synapses.

Morphological Comparison among IVCr, FQF, and PF-DH
The ECS that were obtained with IVCr were lucking ischemia and were observed to be bigger than those obtained from freezing method. But the results were smaller than those obtained from ischemia after 30 seconds and also smaller than those obtained from IVCr after synaptic clefts.

Purpose of Experiment
Some of the important Hemodynamic factors in a kidney are blood pressure and blood flow. They determine the native morphology of glomerular blood capillary loops in kidneys of a living mouse. The purpose of this experiment therefore is to examine the topographical changes in serum proteins, immunoglobulin and albumin passing through the capillary loops (glomerular) and how these proteins are taken back into the renal proximal tubules using immunohistochemistry and in vivo cryotechnique.

Technique and Procedure
The experiment method in use starts with the use of anesthesia on twelve to twenty gram adult mice using sodium pentobarbital. The second step involves opening of the abdominal cavity of four mice. The detection of left kidneys is done under normal circulation of blood. This group of mice is used as a control group. The next group of four mice is used to study glomerular leakage and the following reabsorption of the serum proteins in the renal tubules. For the purpose of use in this step, a mouse with acute renal hypertension is used. The mouse is prepared by the way of ligation of abdominal aorta. In vivo cryotechnique is performed using different blood flows.

Conclusions Based on Experiment Findings

HE staining with IVCT
The renal interstitium between the normotensive tubules had a wide open peritubular space in the normal state. Some Lumina spaces found in the proximal tubules in vivo were however not visible as they were too narrow. The bowmans space and the luminal spaces became wider in the renal cortices.

Immunolocalization of Albumin and IgG in Renal Proximal Tubules
The process revealed immunoreaction products of both IgG and albumin in the blood vessels and at interstitium in renal tubules. Albumin Immunolocalization was partially found in apical parts of renal tubules during the normotensive condition.

Immunolocalization of Immunoglobulin Light or Heavy Chain in Proximal Tubules
The Lambda and the Kappa light chains immunolabelling became easy to detect at hypertensive condition at the apical cell membrane of most proximal tubules. The antibody against IgG-1 heavy chain was however not immunoreacted in the proximal tubules but only in interstitium and peritubular capillaries.

Immunolocalization of albumin and IgG by conventional fixation
Both albumin and IgG were immunolocalized in the bowman spaces and also along apical membranes of the cell of the proximal tubules. The glomerular blood vessels were also partially washed and slightly immunostained due to fixation of perfusion.

Conclusions Drawn from Results of Experiment
The effects of Hemodynamic factors in a kidney in different conditions were observed. These factors have been identified among those that affect the abnormal leakage of the serum proteins, a situation which leads to proteinuria in pathological and experimental state in animal kidneys. In such a case, more proteins are reabsorbed in the renal proximal tubules. The above experiments have helped in establishing that the present in vivo cryotechnique can provide stopped images of functional renal tissues in a living mouse as well as distribution of filtered serum proteins and distribution in the renal proximal tubules.

Global Health Ethics and Science

Global health ethics and is an elaboration of how scientist especially biologists contribute to the integrity of science in the public. A biologist is a scientist devoted to and producing results in biological fields through the study of life. They study organisms and their relationships to their environment. They majorly involve themselves in basic research and they try to invent underling theories that control how organisms work. Most a time, a biologist tries to develop and improve medical, agricultural and industrial processes in diverse fields.

Scientists have contributed a lot to the integrity of science in the public. Particularly, biologists have tried to make life so simple through their several researches that they engage themselves in. Most important of all, our lives rely on these biologists because it is them who find the diseases that affect our lives and further still they try and identify the possible cures to them. Scientists most a time conduct their researches in laboratories with the help of wide variety of other relevant equipments. Some further still use animals and other organisms including plants in their experiments. This mostly happens to botanists, zoologists, and physiologists. Other researches can also take place outside the laboratories when it is necessary. Scientists often work in teams, interacting with engineers, scientists of other disciplines, business managers and even technicians. Examples of biologists include zoologists, botanists, microbiologists, biophysicists, ecologists and computational biologists among others.

In our contemporary societies, scientists and biologists in particular, provide much knowledge needed for progress in technology and medicine. Preferably, we provide the knowledge and understanding needed to make good social decisions on the issues like how to avoid terrible damage our environment or how to deal with epidemics. Certainly, there are mistakes everywhere but a biologist tries as much possible to ensure his or her work is not used for destructive purposes.

Biological reasoning is developed out of philosophy with the need for understanding how things around us work. Scientists are often persons who want to see change taking place in all that they do. They want to know why things happen the way they do. Biologists have the will to explore and discover. They use their resources to bring about change, whether it is a change in medicine, improvements in the environment or creating more efficient recourses. The role of biomedical scientists in an area such as surgery includes tests for emergency and other infections. The successful performance of this key role in modern healthcare relies on the accuracy and efficiency of work by biomedical scientists because patients lives and the treatment of illness depend on their skill and knowledge.

Biologists have helped a great deal in the informal education sector, for instance, in the science centers. Biologists have participated on the boards of science centers, planetariums, environmental centers and even museums to review science contents of scripts for science exhibits, planetarium shows or even environmental programs. They have also involved in giving talks to science centers. Further still, they have collaborated in the creation of museums for science exhibitions and planetarium shows. Biologists have served as science co-coordinators for even scouts troops. This gives explanations of how scientists, particularly biologists, have helped to the integrity of science to the public.

Most scientists have participated in some form of communication of research to non specialist audiences in the past years and this has greatly contributed to the integrity of science in different parts of the world. The research findings have been of great help to both the scholars and even science tutors. Despite the fact that these scientists do not have well communicative skills to educate the public about the social and ethical implications of their researches, they have still managed to encourage institutions to use the research findings that help them in different fields. Biologists support hospital blood banks and the transfusion service. They prepare blood transfusions and plasma fractions to administer to patients and are responsible for ensuring that the blood groups of both donors and patients are compatible. With this knowledge, the biologists have helped the nurses and doctors in various hospitals of the world to manage their professions with keen ness.

Biologists have helped speak out in support of professional development opportunities for teachers of various fields. This has been enabled through a one on one talk between a biologist and a teacher, or even through presentations in teacher workshops or some aspect of science. This contributes to the integrity of the science by implementing the curriculum of the teacher, or even by hiring the teacher as an intern in the science research institution. Biologists have further helped integrate science knowledge in schools of education as they promote the subjects that are science related. This has been achieved by teaching a science course or workshop segment for pre-service teachers. Further still, the biologists have collaborated with education faculty to improve courses on teaching science (Michael, 65). This has led to development of more science courses in the teaching curriculum for teachers-to-be.

Sciences and biologists in particular have spoken out in professional meetings about the importance and value of science involvement in systemic change. They have further reviewed science standards for science accuracy and the stat framework for science education. They have collaborated on writing and adapting science standards. They have also made the public participate on state boards for adoption of standards, instructional materials or teacher certification.

Biologists have had talks with school students and participated in many science activities with the students. They have helped students in their science research projects and they have mentored the students in their laboratories so as to educate the students on the importance of science to the environment and further discussions with students including the one on one talk have helped in integrating the public on science.

The Painted Lady Butterfly

The most common and the most popular butterfly species which can be found in almost all parts of the world is the Painted Lady butterfly. Its colorful appearance fits its name because of the dots of colors on its wings. An adult wear orange and brown on the upper side of its wings. Its forewing has black on its leading edge with white bar and smaller white spots. Shades of brown and gray mark the underside of its wings and while at rest, four small eyespots can be seen on the hindwing.

Painted Lady butterflies are classified under Lepidoptera, where butterflies and moths belong. They are part of the Nymphalidae family or the brush-footed butterflies. They belong to Genus Vanessa and species cardui. Thus, scientifically, painted lady butterflies are called Vanessa curdui.

Vanessa curdui is commonly known as the thistle butterfly because adults sip thistle nectar. Another common name is cosmopolite or cosmopolitan because of its wide distribution around the world. It lives all-year round in tropical temperature, usually in North and South America, Europe, Asia and Africa, but not in Australia and Antarctica. They do not survive in colder areas but they seldom travel south because of overpopulation. Painted Ladies are smaller than other brush-footed butterflies like the monarchs. They reach 5-6 centimeters in width (from 2 - 2 78 inches or 5.1 - 7.3 cm wingspan).

The Life Cycle of a Painted Lady Butterfly
Painted Lady butterfly, like all other butterflies, undergo a process called complete metamorphosis consists of four stages. The first stage begins with mint green, barrel-shaped eggs, usually the size of a pin head, laid singly on the leaves of host plants, and hatch in 3-5 days. Inside each egg is a yellow-striped, brown-green spiny caterpillar which builds a silky, webbed nest, usually in thistle.

The second stage is when the egg becomes a larva. The caterpillar starts to eat using its strong jaws to help its skin get tighter. This tight skin will soon be shed and a new skin emerges, in a process called instar. The caterpillar has five instars over 12-18 days until the caterpillar is full grown usually almost 2 inches long.

Painted Lady caterpillars change their appearance with each instars. This is the stage in the life cycle of an insect between two successive molts. They appear worm-like, with light gray bodies and a darker, bulbous head in early instars but as they mature, they develop noticeable spines with a dark body mottled with white and orange markings. The final instar retains the spines but has a lighter color.

The third stage is when metamorphosis begins. At this stage, the caterpillar rests in a pupa up to 10 days. The caterpillar starts to spin a silk pad using the silk thread that comes out of a hole just below its mouth (spinneret). In this pad, the caterpillar hangs itself. After some time the skin of the caterpillar split open from the head to abdomen revealing a shiny green case underneath called the chrysalis. Inside the chrysalis, the caterpillar becomes liquid reforming itself into a butterfly. From inside, it pushes itself and slowly struggles out, until the case splits open and emerges as a butterfly.

The fourth and the final stage is when the adult butterfly comes out. This happens usually on the second to third week. Butterfly wings are soft and crumpled on its first appearance from the chrysalis. After emerging, it rests first before it unfolds its wings. After a few hours, it is ready to fly. The Painted Lady Butterflys lifespan is only two weeks so it must reproduce and lay eggs during that period to continue the cycle.

Diet, Habitation and Special Adaptation and Defense
The Painted Lady strives on nectars of many plants, especially the composite flowers of the Asteraceae plant family. A variety of hosts plants are good source of the Painted Ladys favorite diet. Caterpillars feed particularly on thistle, mallow, and hollyhock while adult butterflies feed on thistle, aster, cosmos, blazing star, and ironweed.

Like other living organisms, Painted Lady butterflies have special adaptations and defenses. The small caterpillars hide in their silk nests while the colored spots in the adult butterflys wings serve as their best defense against predators because they look very similar to military camouflage.

The Painted Lady enjoys open meadows and fields even in disturbed areas and roadsides. Generally, they inhabit in sunny places where they can find hosts plants and enjoy their favorite nectars.

This species is so quick and easy to rear that is why it is a familiar display in exhibits and projects.

Coprolite deposits reveal megaherbivore moa

The consequences of extinction of Pleistocene megafauna symbolize the origin of plant growth and reproduction traits which have significant implications for interpretations of conservation measures in the contemporary world. The deficiency of dietary data for mega-herbivore species hinders scientific verification of these issues. New Zealand is forms the favorable ecosystem for effective ecological reconstruction. The terrestrial ecosystem in New Zealand was free of mammal species except for the three species of bats and it was dominated by 10 species of avian mega-herbivore.

Moa coprolites potentially provided dietary information for ten moa species. The present vegetation of Central Otago, one of the study areas is highly modified by anthropogenic burning, extensive clearing of land for pastoralism and alien species of grass and weeds. DNA analysis of coprolite revealed that all moa taxa consumed 30 species plants with strong dominance of herbs and subshrubs. This analysis revealed that different moa species in the same habitat fed on similar range of plant species. The relative abundance of different plant types varied slightly within species. Assemblages of seed from coprolite bearing sediment horizons in central Otago provided a proxy for palaeovegetation communities thus providing a peculiar opportunity for assessing plant taxa preferences of moa. The data suggested that herbs and subshrubs formed a larger part of the diet of moa. It further revealed that the moa utilized rockshelters and caves for shelter, roosting and nesting. Some of the variations in plant macrofossil content between individual coprolites were due to seasonal diet variation, with fruitseed being widely available during summer months but less common during winter. Coprolites and gizzards contained plant taxa representing at least eight plant growth forms hypothesized to be responses to moa-browsing and included divarication, toxicity, photosynthetic stems, prostrate-filiramulate habit, fibrous leaves, stinging hairs and low nutrient status, hence confirming that plants with these characteristics were part of moa diet and supported the co-evolution hypotheses. New Zealand has four dryland spring annual herbs including Ceratocephala pungens and Myosurus minimus novae-zelandiae both of which are endangered and lack dispersers.

In conclusion, the coprolite data strongly refute the idea that modern populations of introduced ungulates compete with moa since the coprolites show that moa ate high proportions of plant taxa that are avoided by ungulates and several others that are potentially toxic to mammals. such as e.g. Coriaria Ranunculaceae). In contrast, the data revealed that in some non-forest habitat types, the feeding ecologies of moa are similar to existing ratites that inhabit open habitats.

Diabetes Mellitus as Starving in the Midst of Plenty

(How Diabetes Mellitus is metabolically similar to Starvation)
Diabetes Mellitus is a sugar or glucose related disease in the sense that blood sugar levels (glucose) are abnormally high because the body does not produce enough insulin to maintain and regulate glucose level in the blood.

During the regulation and monitoring of blood glucose level, when levels of blood sugar rise because of the body digesting a certain mealmeals i.e. fed state cells in the pancreas release an hormone called insulin which causes the liver to convert more glucose into glycogen and at the same instance forcing the muscles and fat tissue cells to absorb excess glucose from the blood stream and this in turn decreases regulates blood sugar level.

Blood Sugar level or glucose level in the blood of a human body is tightly regulated and monitored by the cells in the pancreas. If the body glucose level falls dangerously either because of exercise or lack of food for some long period i.e. during unfed state the cells in the pancreas release a hormone glucagons whose effects on liver cells act to increase blood glucose levels by converting glycogen storage into glucose. The glucose is then released into the bloodstream, increasing and regulating blood sugar levels.

The glucose concentration level in the blood stream is known to go high every after a meal is taken. The rise is however brought to control by the insulin hormone which is secreted by the pancreas to stimulate the uptake of glucose into tissues. In a case where there is failure by this insulin mechanism, a diabetic mellitus condition is witnessed. The metabolism condition in Diabetes Mellitus where there is fault of the insulin to regulate and control sugar level arising from meals taken is similar to those in unfed state or starvation in ideology. With little or no meals taken even with the failure of insulin, the glucose in blood level will be under control by the pancreas releasing glucagons a hormone which will induce the liver cells to act and increase blood glucose levels by converting glycogen storage into glucose and release it into the blood stream. That underlines the fact that Diabetic patients however able they are in affording whatever kind of meal, the failure of the insulin mechanism in their body system would dictate special diet a diet which will not increase the blood glucose level and in turn demand insulin secretion to regulate after the meal.


The influence of Drosophila melanogaster on the understanding of basic genetic concepts is examined. Crosses were performed between different species of both wild type and mutant flies. The results of both the first filial generation (F1) and the second filial generation (F2) are presented with the use of Punnett squares. Basic genetic concepts such as autosomal dominance, autosomal recessive, sex-linked dominant and sex-linked recessive characteristics were illustrated.

Gregor Mendel (1822  1884), an Austrian Monk, in the 1980s worked on the heritable traits of plants (pea plants) and revolutionalized the study of genetics. He carefully quantified and analyzed the ways these heritable traits were passed to offsprings. In doing this, he discovered several principles of genetics in use today. However, during the course of his experiments, he attributed the transmission of physical characteristics to heritable factors, which are now known as genes. A gene is said to be the basic unit of genetic information which comprises the DNA.

Drosophila melanogaster is a member of the melanogaster subgroup of the Drosophilidae family. It is commonly known as the fruit fly. This organism is one of the most commonly used species in the study of genetics, mainly because they are easy to handle, they can be bred easily, have relatively short life cycles, have physically evident characteristics which can be used for their gender differentiation, and more importantly, have a relatively simple genome (4 pairs of chromosomes  1 pair of sex chromosomes and 3 pairs of autosomes).

Although Charles W. Woodworth was credited with being the first to breed Drosophila in quantity and for suggesting to W. E. Castle that they might be used for genetic research during his time at Harvard University, it was not until 1910 that Thomas Hunt Morgan began using fruit flies in experimental studies of heredity at Columbia University. Thomas Morgan, some few years after Mendels death used the heritable physical characteristics of the fruit fly (Drosophila melanogaster) to expand the present knowledge and understanding of genetics at that time. He was able to reveal, through his experiments, that the genes that control the transmission of these traits were located on chromosomes. Also, he discovered that certain characteristics (e.g., vestigial versus curly wings) were found on the same chromosomes that also determine their sex.

Gestation period of the fruit fly
Drosophila melanogaster has a complete metamorphosis life cycle with an egg, 3 larval instars, pupa, and adult. They have an average lifespan of about one month (30 days) at an optimal temperature of 29oC (84oF). The time of development of these flies vary with the environmental temperature. As temperatures increase, the developmental time increases also. At temperatures of about 21oC, the gestation period takes an average of two weeks after mating and fertilization of eggs before offsprings mature as adults. The process of development goes through different stages. After fertilization, a worm-like larva emerges from the embryo after about 24 hours. The larva feeds on microorganisms and also on the decaying material which surrounds it, and continues to grow until it molts three times (first, second, and the third instar). The molting process occurs one, two and four days, respectively after hatching to produce a third instar larva which molts again one more time, forming a pupa. The pupa is usually immobile and its body is modified and developed to form a winged adult.

After emerging from the pupal shell, the adults are ripe enough for mating within 8 to 12 hours and in most cases can survive under laboratory conditions for over a month. Two days after mating, the females begin to lay eggs and within three to four days after molting, the first instar larva becomes active.

Characteristics of the fruit fly (Wild type)
The wild type fruit fly, which possesses the normal phenotype, has distinguishing characteristics. Both the male and female wild type flies have similarities and differences also. Usually, the older male flies have darker posterior abdominal parts when compared to the females which have lighter parts. Another distinguishing characteristic is the abdominal tip. The abdominal tip is more round in the males than in the females. Also, male wild type flies are considerably smaller than the females. However, this cannot be used as a reliable distinguishing character. The most reliable distinguishing factor is the presence or absence of sex combs (short dark bristles on the distal part of the first pair of legs). Sex combs are present in all male wild type flies, regardless of the age or level of maturity.

Characteristics of the fruit fly (Mutant)
Mutations in genes that control the phenotypic characteristics of the flies produce mutant flies. Mutant flies are phenotypically different from the wild type flies. A mutant fly is said to be a fly that deviates from the normal phenotypic characteristics of the wild type fly. The specific mutation of the fly is designated with a letter(s) appropriate for the specific phenotype of the mutation. Dominantly inherited mutations are recorded in upper case alphabets while recessively inherited mutations are represented with lower case alphabets. Sex-linked mutations are designated as superscripts to the corresponding X- or Y- chromosome.

The fruit fly, Drosophila melanogaster, has been used for several years to demonstrate Mendelian genetics. According to Mendel, some genetic traits are dominant, in the sense that, they are always expressed in heterozygous conditions, while others are recessive. Recessive traits are not expressed in heterozygous conditions. An organism is said to be homozygous for a particular trait if it expresses two identical alleles for the same trait, while it is said to be heterozygous if it has two different alleles for the same trait.
Crosses between different flies with either the homozygous allele or heterozygous allele confirm Mendels theories. The common phenotypes and notations of the wild type and mutant flies are as follows

Table 1. Abbreviations and descriptions of various common phenotypes. Source Mendelian Genetics Lessons from the Fruit Fly, 2009
Wild typeMutantEye color  Red ()White (w), Brown (se)Eye shapeNormal ()Lobed (L), Bar (B)AntennaeNormal ()Spineless (ssa)Wings Normal ()Vestigial (vg), Apterous (ap), DumpyJagged (dp)Body colorNormal ()Yellow (y), EbonyDark brown (e)BristlesNormal ()Singed (sn), Spineless (ss)

1. Cross between a light body male and a wild type female
The light bodied male will be designated (y), while the wild type female will be designated (). This cross is a monohybrid cross because it only involves a pair of alleles. This cross will pair a homozygous recessive (yy) male with a homozygous dominant () wild type female. The male will produce haploid sperms carrying the (y) allele while the eggs will carry the () allele. The first filial generation (F1) offsprings will all be heterozygous (y) for normal body colour. This is illustrated in the Punnett square below.

EggSpermyYyyyyFigure 1. Punnett square showing the genotypes of the P-generation and the expected genotypes of the F1 generation.

EggSpermyyyyyyFigure 2. Punnett square showing the genotypes of the F1-generation and the expected genotypes of the F2 generation.

A cross between the members of the first filial generation is shown in Fig. 2. The Punnett square reveals a genotypic ratio of 1 homozygous wild type, 2 heterozygous normal bodied, and 1 homozygous recessive light bodied. The phenotypic ratio is 1 light bodied and 3 normal bodied flies.

2. A cross between a light bodied female and a wild type male
Just like the cross illustrated in Fig. 1, the F1 generation offsprings would all be heterozygous (y) for normal body colour. The F2 generation offsprings will have a phenotypic ratio of 1 light bodied and 3 nornal bodied flies, and a genotypic ratio of 1 homozygous wild type, 2 heterozygous normal bodied, and 1 homozygous recessive light bodied.

3. A cross between a jagged winged female and a wild type male
The jagged winged female will be designated (dp), while the normal wild type male will have a notation of (). During meiotic division, haploid gametes are produced, with the male producing () sperms and the female producing (dp) eggs. The results of this cross is illustrated in the Punnett square below.

EggSpermdpdpdpdpdpdpFigure 3. Punnett square showing the genotypes of the P-generation and the expected genotypes of the F1 generation.

EggSpermdpdpdpdpdpdp Figure 4. Punnett square showing the genotypes of the F1-generation and the expected genotypes of the F2 generation.

Fig. 3 shows that all the offsprings of the first filial generation are heterozygous for normal wings. However, a second cross between the members of the first filial generation by mating a F1 male (normal) with a F1 female (normal) reveals a phenotypic ratio of 1 jagged winged and 3 normal winged flies. The genotypic ratio is 1 homozygous dominant normal, 2 heterozygous normal, and 1 homozygous recessive jagged winged fly. This is evident in Fig. 4.

4. A cross between a jagged winged male and a wild female
The notation for this cross is as the same for the cross done in Fig. 3 and 4. The first generation offsprings will all be heterozygous for normal wing. However, a second intra-F1 generation cross yields 3 normal wild type and 1 jagged winged phenotypes. The genotypic ratio is 1 homozygous wild type 2 heterozygous wild types 1 homozygous jagged.

5. A cross between a dark bodied male and a wild female
The dark bodied male will be designated (e) while the wild type female will be designated (). The Punnett square below reveals the predicted outcomes of the cross.

EggSpermeeeeeeFigure 5. Punnett square showing the genotypes of the P-generation and the expected genotypes of the F1 generation.

EggSpermeeeeeeFigure 6. Punnett square showing the genotypes of the F1-generation and the expected genotypes of the F2 generation.

The parents will produce a F1 generation in which all the offsprings are heterozygous wild type. A second cross between members of the F1 generation reveals a phenotypic ratio of 3 normal wild type 1 dark bodied. The genotypic ratio will then be 1 homozygous wild type 2 heterozygous wild types 1 homozygous dark bodied, as shown in Fig. 6.

6. A cross between a dark bodied female and a wild type male
Using the same notation as above, the dark bodied female is represented by (e) and the wild type male is represented by (). The male produces haploid sperms carrying the () allele, while the female also produces eggs carrying the (e) allele. A cross between these gametes will produce offsprings which are all heterozygous wild types. This represents the F1 generation (Fig 5). A cross between members of the F1 generation will produce two phenotypically different groups of offsprings. The offsprings produced are either wild type or dark bodied in a ratio of 31 respectively. The genotypic ratio is 1 homozygous wild type 2 heterozygous wild types 1 homozygous dark bodied.

7.  A cross between a brown eyed male and a wild type female
The genetic notation is a bit different from the notation of the other traits. This is because the trait for eye color is controlled by a sex-linked gene (Peebles, Whitmarsh,  Burnham, 2001). Therefore, male fly with brown eyes will be designated XseY while the female wild type will be designated XX. The male will produce haploid sperms with either Xse or Y, while the female will produce haploid eggs with X.

EggSpermXseYXX XseX  YXX  XseX  YFigure 7. Punnett square showing the genotypes of the P-generation and the expected genotypes of the F1 generation in a X-linked cross.

EggSpermXYXX  XXYXseX  XseXse YFigure 8. Punnett square showing the genotypes of the F1-generation and the expected genotypes of the F2 generation in a X-linked cross.

The above illustrated crosses involve sex-linked or X-linked characteristics. The first cross between the parents produced two kinds of genotypes, that is, X Xse and X  Y. This interpreted means that there are two wild type females and two wild type males, despite the fact that one of the parents was brown eyed. In the second cross illustrated in Fig 8, there are four genotypes and three phenotypes. The genotypic ratio is 1111 all through. Half of the males are wild types while the other half has brown eyes. Similarly, one half of the females are also wild types.  What if the phenotypes of the P1 generation are reversed

8. A cross between a brown eye female and a wild type male
The cross is demonstrated with the Punnett square in Fig. 9. The notation used above is still repeated, with the wild type male designated XY and the brown eyed female designated by XseXse.

EggSpermXYXseX XseXse  YXseX  XseXse  YFigure 9. Punnett square showing the genotypes of the P-generation and the expected genotypes of the F1 generation in a X-linked cross.

Again, the offsprings of this cross are an opposite of the P1 generation. The offsprings with the Y notation are the males while the others lacking the Y are females. Crossing these offsprings will yield the following

EggSpermXseYXX  XseXYXseXse  XseXse YFigure 10. Punnett square showing the genotypes of the F1-generation and the expected genotypes of the F2 generation in a X-linked cross.

Half of the F2 males are expected to be wild type, while the other half should have brown eyes. The F2 females are also expected to have a 1 wild type 1 brown eyed phenotypic ratio.

Although there were no brown eyed males in the parental generation, this does not discredit the result that there were both wild type and brown eyed males in the F2 generation. Also, examining the results of the F2 generation, heterozygous wild types can also be found.

Mendel, in the process of conducting several of his scientific experiments, observed the consistent appearance of some traits, which he called dominant, and the traits that reappeared recessive. The dominant traits are always expressed, no matter the number of alleles present (either one or two). This is demonstrated by the crosses in Figs. 1  7. The recessive alleles can only be expressed when both alleles are present. The inheritances of these traits abide by the Mendelian rules for characters having an autosomal dominant-recessive relationship.

Reciprocal crosses provide a means of distinguishing between autosomal traits and sex-linked characters. A reciprocal cross is performed by switching the phenotype of the parent in a direct relationship to its sex in the P1 generation. The fact that when reciprocal crosses were conducted in Figs. 1 -7 shows that the characters been inherited are not sex-linked. This makes them autosomal, and the dominant alleles are referred to as autosomal dominant while the recessive alleles are referred to as autosomal recessive. Reciprocal crosses have no effect on the F1 and F2 results for an autosomal trait like wing-type, but it does for a sex-linked trait like eye color.

X-linked characters can also be either dominant or recessive. There can never be any Y-linked dominant or recessive character. This is because the only major characteristic that may be located on the Y-chromosome are those that determine male differentiation. A male fly is said to be hemizygous because it can only possibly carry one copy of a gene on its X-chromosome, with the Y-chromosome being relatively redundant. Female flies with dominant wild type genes are asid to be X- linked dominant and those with the recessive genes are said to be X-linked recessive. This is because they carry two copies of the X-chromosome, unlike the males who have only one copy. A mutant fly must have the mutation on the single copy of its X-chromosome. In a mutant female fruit fly, the mutation must occur on both copies before it can be said to be mutant (X-linked recessive).

The use of Tamoxifen for the treatment of breast cancer

As indicated by Morrow and Gradishar (2002), breast cancer affects women in the ratio of one to eight in their lives that is approximately 12 of all women. It is the second major cause of cancer deaths among women in the world exceeded only by cancer of the lungs. The chance that the death of a woman will result from breast cancer is about 3. Though one cannot clearly tell why some women get breast cancer there are some risk factors associated with its development. Age, genetics and personal factors, such as early adolescence and late menopause, are all linked to breast cancer development. Breast cancer also affects males though not at a high level as compared to women. Breast cancer develops in men who have attained the age of 60 years. It is estimated that around 2,000 new cases of invasive breast cancer are diagnosed in men and that about 450 of them die annually. Breast cancer cases in men comprise about 1 of all cases of breast cancer.

Breast cancers can be treated through various ways. One of them is through drugs.  Tamoxifen is a medication taken through the mouth as a tablet, which works through interference of the activity of estrogen a female hormone that promotes development of breast cancer. This drug has been in use for treatment of breast cancer for both men and women for more than 30 years. It has been used for treating both early-stage and breast cancer that has metastasized.

Tamoxifen is also used as an adjuvant therapy to avoid initial breast cancer from recurring and also help reduce cases of emergence of new cancers in the breast. When used for treatment of metastasized breast cancer, Tamoxifen impedes the growth of cancerous cells that may be present in the body. For the past one decade, Tamoxifen has been used to lower the risk of breast cancer among women. Women with ductal carcinoma in situ (DCIS), a condition that leads to the development of breast cancer are treated using Tamoxifen. The advantages of using Tamoxifen in the treatment of breast cancer outweigh its disadvantages by far. The period through which patients with breast cancer should take Tamoxifen varies from one patient to another. This variation occurs as a result of the cancer cells response to treatment and also other factors.

Estrogen is one of the major factors that promote the development of breast cancer cells. Some breast cancers are referred to as estrogen-receptor positive because they have a biding site for estrogen. Breast cancer cells require estrogen so as to grow. The effect of estrogen on breast cancer cells is greatly impeded by Tamoxifen making it to be referred to as anti-estrogen.  Tamoxifen competitively binds to estrogen receptors on breast cancer cells, thereby developing a nuclear complex that lowers the synthesis of DNA. As a result it hinders the expression of estrogen-regulated genes, which include the growth as well as angiogenic factors produced by the cancer cells that may activate growth by autocrine mechanisms. This leads to a blockage in the G1 phase of the cell cycle thereby slowing down proliferation of the cells. Tamoxifen makes caner cells accumulate in the G0 and G1 stages. Tamoxifen is therefore cytostatic. As a result breast cancer regresses due to altered balance between cell loss and cell proliferation. The drug may also directly bring about programmed cell death. Tamoxifen is effectively absorbed in the mouth. It is then distributed in the body through the blood. High concentrations of the drug are found in the uterus, breast, and the ovary. Its metabolism occurs through the hepatic microsomal oxidation system.

The rate of death due to breast cancer, according to Klinik, et al (1998), has been declining since 1990 due to increased awareness and screening. Chemotherapy is one of the novel techniques used in the treatment of breast cancer. During the last few years hormonal treatment of breast cancer has been revisited. Future research trends are aimed at treating breast cancer based on endocrine perspective.

Role of Micronutrients on Enzyme Activity

All biological activities are mediated by enzyme activities. Enzymes are specific to their substrates and function at the optimum levels under different conditions of temperature, pH, and many times also in the presence of specific coenzymes, cofactors and a metal ion. The metal ion is converted to oxidized andor reduced state by the change of electron(s) during a biochemical reaction.

In the present experiment Pyruvate the end product was estimated in the presence its substrate and as effected by four different concentrations (0.2 mg cl 0.4 mg cl 0.5 mg cl and 0.8  mg cl) of magnesium chloride. The enzyme activity was assayed at 30 min intervals for 180 minutes.

It was observed that addition of up to 0.4 mg cl of magnesium chloride concentration the activity increased in a linear fashion even at the initial stage of the reaction (Fig 2). But at higher concentrations, 0.6 and 0.8 mg cl the enzyme activity steeply falls down and was seem to stabilize around that concentration.

It can also be seen in Fig 1 that at the same increasing concentrations of magnesium chloride, the enzyme activity proceed in a linear increasing manner till 180 minutes (Tubes 4 and 5 Fig 1 and to some extent in tube 2 (Fig 1). But in tubes 3 and 6 the activity increase is not that comparably steep.

Many enzymes need cofactors which are non-protein molecules which function by binding to show activity. Many metal ions such as iron in reduction - oxidation reactions, magnesium, copper and many more are involved actively in such reactions.

In addition to positive intimate involvements of metal ions in many biochemical reactions met als can also be strong inhibitors of enzymes. They also may be required for the start of the reaction as in the present experiments. But beyond certain concentrations of their ions their roles may become passivive, non-active or strongly inhibitory.

In the present concentrations the linear incremental trend of the enzyme up to 04 mg cl concentrations of magnesium ions was observed till the ned of the experiment in tubes 4 and 5. But in other tubes after 120 minutes magnesium is either inhibitory or is not required. It is less likely to be inhibitory because the activity proceeds without dropping albeit slow.

The present experiments reveal a trend of magnesium ions for enzyme activity with PNP as a substrate. More experiments need for obtaining specific information.

Analysis and Discussion of Available Genomic Tools for Obligate Inbreeding and Obligate Outcrossing Rhabiditic Species

Evolution of Genetics
During the nineteenth century may philosopherresearches forced their imaginations to conceptualize the variability in all organisms. Charles Darwin was the frontrunner and based on his extensive travels and studying all kinds of life forms, even fossils of plants and animals.

The Variation of Animals and Plants under Domestication The8 Variat ion of Animals and Plants under Domestication (1868) proposed a hypothetical mechanism of heredity as a provisional hypothesis called pangenesis. In the theory of evolution (pangenesis) of Hugo de Vries 1988 term for the same concept) he referred to heritability of character called the pangene whose transmission occurs from parent to offspring. The theory contained many facts but sadly without reasoning and contained explanations to phenomena such as atavisms, intermediate nature of hybrids, use and disuse of Lamarck, limb regeneration etc. He lacked imagination on a clear mechanism of inheritance. That he had the foresight to use the principle of inheritance to explain variability without any clues on meiosis, chromosomes or for that matter, much less genes, DNA, or the basic principles of Mendelian genetics such as random segregation and independent assortment is significant (to be discussed later).

Darwin even did simple crosses with snapdragons but based the results on Lamarcks theories. What is surprising is that Darwin, in his thinking years (1835-1870), paid absolutely no attention to the cell and developmental biology and genetics that was beginning to happen at the same time in Europe. Ref

Around the same time, in 1866 Mendel published results of his work from his classical work on pea plants. Mendel used peas based the knowledge that they were bisexual with both cross and self-pollinating mechanisms with many known traits and had short life span. He followed a scientific approach to make clear controlled cross pollinations to control the characters. He followed these very characters in the progenies to look for their inheritance and distributions and ensured that data was accurately collected and analyzed statistically, and finally presented data with inferences on the two laws of heredity to Versuche ber Pflanzen-Hybriden in the Verhandlungen des Naturforschenden Vereins zu Brnn, following two lectures he gave on the work in early 1865. The highlights of the work are there is a pair of contrasting (dominant and recessive alleles) characters which segregate independently and get assorted randomly but in certain definite proportions in progenies depending on whether they are dominant or recessive and which are predictable.

The term gene was coined by a Danish Botanist Wilhelm Johannsen in 1909 for these units of heredity. Genetics was named by William Bateson in 1905 for the study of the Science of Heredity.

A mention must be made that Mendel in his presentation acknowledged richly the contributions of his contemporaries as follows inexhaustible perseverance of Klreuter, Grtner, Herbert, Lecoq, Wichura and others, Mendel further observed that .so far, no generally applicable law governing the formation and development of hybrids has been successfully formulated.

He also exclaimed that none of these researchers made any effort to enquire on determining the number of different groups to which different progenies can belong and also to classify them according to different generations as also on the statistical inferences. He wrote, none has been carried out to such an extent and in such a way as to make it possible to determine the number of different forms under which the offspring of the hybrids appear, or to arrange these forms with certainty according to their separate  generations, or definitely to ascertain their statistical relations.

The true significance of Mendels work had to wait till his paper  was rediscovered by De Vries, Correns and Tschermak 40 years after its publication.

Ever since the principles of inheritance and variation were re-established Genetics, coined by Bateson has become the mainstay for more experimental approaches to creating variability and ensuring heritability of desired characters from parents to offspring in crop improvements. The principles of inheritance were originally analyzed and inferred from painstaking but clear cross pollinations in pea plants for many years. The Mendelian principles proposed that discrete units of characters, factors (now called genes), were inherited by offspring from parents. They are assorted and segregated independently. Mendel discovered all this without any idea of Genes genes, Meiosis meiosis whose roles in heredity were elaborated years after his death and which showed how Mendels laws are carried out. This stimulated considerable researches on verifying these principles.  Drosophila melanogaster Drosophila melanogaster attracted maximum attention for applications of Mendelian genetics. Morgan established the Mendelian-Chromosome theory of Heredity.

The Gene was thus born. It is not clear when exactly the term gene was coined but thie word was used in the literature. But (website o the The National Human Genome Research Institute, NIH, USA) describes that in 1869, in a hospital Johann Friedrich Miescher isolated a substance called nuclein from a pus of a wound. Later this substance came to be known as nucleic acid. In 1879 Walter Flemming observed mitosis in salamander embryos developed stain to stain chromosomes clearly and described chromosome behavior during mitotic division.

By 1900 Mendels concepts (factors) were getting nearly understood when three European Botanists - Carl Correns, Hugo DeVries, and Erich von Tschermak, unaware of each other, independently reviewed the literature before publishing their own respective results on the laws of inheritance from their experiments of pea plants (1902). In 1905 a Mendels supporter, William Bateson felt the need for a new word genetics is supposed to have suggested so in letter before W. Johanssen coined the word gene as well as phenotype and genotype (1909 cited in ..  Roberts, H. F. Plant Hybridization before Mendel. Princeton Princeton University Press, 1929 and later It is significant to mention that in 1902 itsel the Alkaptoneuria disease also was observed to inherited as per Mendels laws Garod, W, cited in This year (1902) Suttons observations from grasshopper cells confirmed Theodor Boveris earlier observations (during 1880s and 1890s) that  chromosome numbers are reduced to half the original numbers as egg cells mature and also that  the segregation pattern of chromosomes during meiosis matched the segregation patterns of Mendels genes.    This also important support to the chromosomal theory of heredity and further support was obtained from .experimental observations of Thomas Hunt Morgan, Alfred Sturtevant, Hermann Muller and Calvin Bridges that genes are arranged in a linear waay on chromosomes and that they are linked. In 1911 in fruitflies.

In 1941 working on x-irradiated Neurospora crassa Beadle and Tatum found that genes mutate and cause biochemical deficiencies making it imperative for the organism to to grow on an artificial medium only when supplemented with a particular nutrient, an amino acid in their example. Their proposal further clarified the understanding of the gene as a functional unit which governs the formation of only one enzyme.

Earlier in 1928 Griffith, working on  two strains of Streptococcus pneumonia,  harmless (R) and virulent (S) observed that the R strain could get converted into harmful deadly S strain by simply injecting simultaneously the R strain as well as the heat killed S strain into mice. The mice though injected with killed S strain was not unexpected to develop the pneumonia disease but they did. Such results were observed for a long time in Oswald Averys lab till 1940s. In 1941 Avery along with
A gene as broadly known today is a unit of heredity in a living or dead organism with a predetermined function functioning in a synchronous manner in synchrony with millions of other genes in the total primary genetic configuration of an organism. It is a strand of a unique DNA structure with a code for ordering production of a protein and RNA for a specific function. This crisp short description of a gene has been facilitated by many scientists working on different directions simultaneously and sequentially. Every organism has millions of genes arranged in a linear fashion in the whole complement called the genome.

The understanding of gene was a concept in the Classical Era described so far. A gene as broadly known today is a unit of heredity in a living or dead organism with a predetermined functioning in a synchronous manner in synchrony with millions of other genes in the total primary genetic configuration of an organism. It is a strand of a unique DNA structure with a code for ordering production of a protein and RNA for a specific function. This crisp short description of a gene has been facilitated by many scientists working on different directions simultaneously and sequentially. Every organism has millions of genes arranged in a linear fashion in the whole complement called the genome.

The broad area of Genetics has proliferated quantitatively and ramified qualitatively into many other disciplines. The total evolution of Genetics may be considered in different eras.

In the Classical described thus far the attempts have repeatedly been made to dexcribe gene as a structural and a functional unit with no references to possibilities of architectural diversities. In the following discussions an idea of how different this concept can be is discussed.

Watson and Crick
J. D. Watson was a molecular biologist and Framcis Crick was a molecular biologist, physicist and a neuroscientist, are the codiscoverers of the DNA structure working the the Cavendish Laboratory,   . The first announcement of the DNA structure was made by the Director of the Cavendish Laboratory, Sir Lawrence Bragg at Solway Conference on proteins in Belgium in 1953. Watson and Crick published this paper in Nature 1953.

They acknowledge Pauling and Coreys paper made available to them prior to its publication and found some objections to the observations made by them. They also had on hand a paper in press by Fraser. Again Watson and cricks convictions about their own model was strong enough to add their comments in their paper in Nature (1953).

According to Watson and Crick the DNA has two helical chains each coiled around the same axis. Based on normal chemical assumptions the two chains but not their bases are related by a dyad perpendicular to the fibre axis. Both chains follow right handed helices but due to the dyad, the sequences of the atoms in the two chains run in opposite directions.

They were fully aware of the then available information from other active researchers on nucleic acids (from accessexcelllence)
DNA is made up of subunits which scientists called nucleotides.
Each nucleotide is made up of a sugar, a phosphate and a base.
There are 4 different bases in a DNA molecule
Adenine (a purine)
Cytosine (a pyrimidine)
Guanine (a purine)
Thymine (a pyrimidine)
The number of purine bases equals the number of pyrimidine bases
The number of adenine bases equals the number of thymine bases
The number of guanine bases equals the number of cytosine bases

The basic structure of the DNA molecule is helical, with the bases being stacked on top of each other

They both made good use of their expertise as well some additional information obtained informally. It was primarily based on the x-ray crystallography data from Rosalynd Franklin and Maurice Wilkins.

Working with nucleotide models made of wire, Watson and Crick attempted to put together the puzzle of DNA structure in such a way that their model would account for the variety of facts that they knew described the molecule. Once satisfied with their model, they published their hypothesis, entitled Molecular Structure of Nucleic Acids A Structure for Deoxyribose Nucleic Acid in the British journal Nature (April 25, 1953. volume 171737-738.) It is interesting to note that this paper has been cited over 800 times since its first appearance

Kary Banks Mullis and Polymerase Chain Reaction
Kary Banks Mullis is a self-motivating achiever biochemist with broad and diverse and even unrelated interests but with a strong sense of adventure and attitude of perseverance to pursue the convictions of imaginations till achieved. He is a well known popularly as intellectual maverick and a Chemistry Nobel Laureate for his contributions to the development of Polymerase Chain Reaction (PCR) in1993. In fact the idea for this most popular technology was originally available in a paper by Kleppes et al (1971). Kleppes et al.(1971) described an in vitro enzymatic method to replicate a short DNA template with primers.

But the significance of this idea was best recognized only by Kary Mullis in 1986.  While working on the idea of using DNA polymerase to copy a desired DNA sequence by using two primers to bracket it. In his imagination this technique was expected to facilitate copying small strand of DNA almost an infinite number of times.

One of the complications of this was that high heat used at the beginning of the experiment destroyed the DNA polymerase. Logically he turned his attention to the DNA polymerase enzyme from Thermophilus aquaticus Thermus aquaticus. Taq polymerase  is a thermostable  DNA polymerase DNA polymerase named after Thermophilic thermophilic bacterium  Thermus aquaticus Thermus aquaticus which it was originally isolated by Thomas D. Brock Thomas D. Brock in 1965.

T. aquaticus is a  Bacterium bacterium found in the Hot springs hot springs and Hydrothermal vent hydrothermal vents, and Taq polymerase was identified as an  Enzyme enzyme able to withstand the protein-denaturing conditions (high temperature, temperature optimum 80C) required during PCR. DNA polymerase was first isolated from Thermus aquaticus in 1976. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus by A Chien, D B Edgar, and J M Trela in Journal of Bacteriology 127 (3) 15501557.) The first advantage that was found for this thermostable (temperature optimum 80C) DNA polymerase was that it could be isolated in a purer form (free of other enzyme contaminants) than could the DNA polymerase from other sources. Subsequently T. aquaticus was the source of many other enzymes such as Taq I restriction enzyme, aldolase, dehydrogenases, alkaline phosphatases RNA polymerases, DNA ligase DNA ligase,  Alkaline phosphatase Alkaline Phosphatase,  Oxidase NADH Oxidase,  Isocitrate dehydrogenase Isocitrate Dehydrogenase,  Maltase Amylomaltase, and the ever-popular  Lactate dehydrogenase Fructose 1,6-Bisphosphate-Dependent L-Lactate Dehydrogenase  all of which have properties of tolerating biologically inactivating high temperatures. Such researches also stimulated great interest in looking for enzymes in many other thermophilic organisms. One such organism is Pyrococcus furiosus Pyrococcus furiosus whose polymerase called Pfu was compared with Taq DNA polymerase and found that Pfus superior thermostability and proofreading properties compared to thermostable Taq polymerases as well as those of other organisms. Similarly Pfus thermostability and proofreading properties were superior compared to other thermostable polymerases.. Pfu DNA polymerase possesses 3 to 5  Exonuclease exonuclease proofreading activity, meaning that it works its way along the DNA from the  5 end 5 end to 3 end 3 end and corrects Nucleotide nucleotide-misincorporation errors. This implies that PCR fragments  generated by Pfu DNA polymerase is likely to show fewer errors than those from Taq-polymerase inserts. Consequently Pfu is more commonly used for molecular cloning of PCR fragments than the historically popular Taq.

Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6 mutated products when amplifying 1kb fragments using PCR. However, Pfu is slower and typically requires 12 minutes per cycle to amplify 1kb of DNA at 72 C. Using Pfu DNA polymerase in PCR reactions also results in blunt-ended PCR products.

Pfu DNA polymerase is hence superior for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity

Polymerase Chain Reaction
Polymearse Chain Reaction - Technique
This reaction has become well integrated into biology and medicine investigations. Most of the applications of this are in amplifying (multiplying) a single or a few copies of DNA (gene) exponentially resulting in very large numbers of copies (thousands to millions of copies) of a particular DNA sequence. The basis of this method is a repeated cyclic process of three differential temperature treatments (heating and cooling) to a reaction mixture containing DNA fragment to be amplified. The different temperatures (heating and cooling) are meant for DNA melting and DNA replication respectively with a DNA polymerase.  Primer (molecular biology) Primers (short DNA fragments) containing sequences complementary to the target region along with a Taq polymerase DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion Chain reaction chain reaction in which the DNA template is Exponential growth exponentially amplified. PCR can be extensively modified to perform a wide array of Genetic engineering genetic manipulations.

The cycling reactions
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.

Denaturation at 94C
1 minute 94 C During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (for example  the extension from a previous cycle).

Annealing at 54C 45 seconds, forward and reverse primers
The primers are jiggling around, caused by the Brownian motion. Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last a little bit longer (primers that fit exactly) and on that little piece of double stranded DNA (template and primer), the polymerase can attach and starts copying the template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.
extension at 72C 2 minutes, only dNTPs

This is the ideal working temperature for the polymerase. The primers, where there are a few bases built in, already have a stronger ionic attraction to the template than the forces breaking these attractions. Primers that are on positions with no exact match, get loose again (because of the higher temperature) and dont give an extension of the fragment. The bases (complementary to the template) are coupled to the primer on the 3 side (the polymerase adds dNTPs from 5 to 3, reading the template from 3 to 5 side, bases are added complementary to the template)

The ladder is a mixture of fragments with known size to compare with the PCR fragments. Notice that the distance between the different fragments of the ladder is logarithmic. Lane 1  PCR fragment is approximately 1850 bases long. Lane 2 and 4  the fragments are approximately 800 bases long. Lane 3  no product is formed, so the PCR failed. Lane 5 multiple bands are formed because one of the primers fits on different places.

Because both strands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the wanted gene before the cycling starts, after one cycle, there will be 2 copies, after two cycles, there will be 4 copies, three cycles will result in 8 copies and so on. After 35th cycle there would be 68 billion (236) copies from one single fragment.

To check whether the PCR generated the anticipated The amplified DNA fragment (o Amplicon amplicon) generated by PCR, the products are checked for trueness to the expectations by agarose gel electrophoresis along with standard marker DNA fragments of known molecular weights. This method provides sizes of the test molecules when compared with the bands of DNA ladder formed

Optimizing PCR
Due to both established variations of this technique and newly developing broad applicability opportunities of this method developed by Kary Mullis, PCR is indispensible for a majority of basic and applied biological investigations. In many situations the technique has to be subjectively standardized before real applications on a case to case basis. As mentioned earlier, although most extensively used, the method has some limitations such as misreading of sequences, its sensitivity to contaminating DNA and erroneous DNA products and others. A number of techniques and procedures have been developed for optimizing PCR conditions  (PCR from problematic templates. Focus 221 p.10 (2000). Helpful tips for PCR. Focus 221 p.12 (2000). Separate prerun of agarose gel without the PCR samples is run as a standard procedure. PCR  set up is maintained in a separate place thoroughly cleaned before and after the electrophoresis runs. Primer design is extremely important for getting good and reliable results.

Since then there have been major innovations made, as applications of PCR have been essential to the progression of certain areas in science. These include the mapping of the human genome project, single sperm analysis, molecular archaeology and ancient DNA, molecular ecology and behaviour, disease diagnosis and drug discovery.

PCR has enabled the analysis of molecules from the past in order to examine species that may now be extinct and to determine their relationships to present day organisms. One example of this analysis is, DNA from a 5000 year old frozen mummy, the Tyrolean ice man, was analysed in a hypervariable region of mitochondrial genome and this late Neolithic individuals mitochondrial type was found in 13 of 1200 people and was closely related to the types determined from central and northern Europe populations.

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Classification of organisms
Molecular archaeology
Mutation detection
Cancer research
Detection of pathogens
DNA fingerprinting
Drug discovery
Genetic matching
Genetic engineering
Pre-natal diagnosis
Mutation screening
 Drug discovery
 Classification of organisms
 Molecular Archaeology
 Molecular Epidemiology
 Molecular Ecology
 Genomic cloning
 Site-directed mutagenesis
 Gene expression studies

Applications of PCR
Basic Research

Mutation screening
 Drug discovery
 Classification of organisms
 Molecular Archaeology
 Molecular Epidemiology
 Molecular Ecology
 Genomic cloning
 Site-directed mutagenesis
 Gene expression studies

Applied Research
Genetic matching
 Detection of pathogens
 Pre-natal diagnosis
 DNA fingerprinting
 Gene therapy

Applications of PCR

Molecular Identification
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
DNA fingerprinting
Classification of organisms
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens
Genomic cloning
Human Genome Project

Genetic Engineering
Site-directed mutagenesis
Gene expression studies

Variations of PCR technique from google

Allele specific PCR This is based on single nucleotide polymorphisms requiring exact prior information on the DNA sequence including allele differences and used both for diagnostics and cloning. But under stringent conditions this less efficient.

Polymerase cycling assembly Assembly PCR or Polymerase Cycling Assembly (PCA) PCR is performed on a pool of long oligonucleotides with short overlapping sequences for artificial synthesis of long synthesis of long oligonucleotde sequences.

Assymmetric PCR It is used in hybridization and sequencing. This amplifies one DNA strand in a double stranded DNA template. One of the two primers needed in excess for the strand directed sequence. This has been further modified to accelerate the slow rate of reaction.

Helicase-dependent amplification Helicase-dependent amplification is the same as the normal  PCR, but uses a constant temperature rather than cycling through denaturation and annealingextension cycles.  DNA helicase DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation. Hot-start PCR (page does not exist) Hot-start PCR a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerases activity at ambient temperature, either by the binding of an Antibody antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-startcold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Main article  Variants of PCR
Allele-specific PCR (page does not exist) Allele-specific PCR a diagnostic or cloning Single-nucleotide polymorphism single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between Allele alleles, and uses primers whose 3 ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence. See SNP genotyping SNP genotyping for more information.

Polymerase cycling assembly Assembly PCR or Polymerase Cycling Assembly (PCA) artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.

Asymmetric PCR (page does not exist) Asymmetric PCR preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in Sequencing sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.

A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.

Helicase-dependent amplification Helicase-dependent amplification similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealingextension cycles.

DNA helicase DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

Hot-start PCR (page does not exist) Hot-start PCR a technique that reduces non-specific amplification during the initial set up stages of the PCR. It may be performed manually by heating the reaction components to the melting temperature (e.g., 95C) before adding the polymerase. Specialized enzyme systems have been developed that inhibit the polymerases activity at ambient temperature, either by the binding of an  Antibody antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-startcold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Intersequence-specific PCR (page does not exist) Intersequence-specific PCR (ISSR) a PCR method for DNA fingerprinting that amplifies regions between simple sequence repeats to produce a unique fingerprint of amplified fragment lengths. Inverse polymerase chain reaction Inverse PCR is commonly used to identify the flanking sequences around Genomic genomic inserts. It involves a series of  Restriction digest DNA digestions and  Self ligation self ligation, resulting in known sequences at either end of the unknown sequence. Ligation-mediated PCR (page does not exist) Ligation-mediated PCR uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers it has been used for DNA sequencing DNA sequencing,  Genome walking genome walking, and  DNA footprinting DNA footprinting.

Methylation-specific PCR Methylation-specific PCR (MSP) developed by Stephen Baylin and Jim Herman at the Johns Hopkins School of Medicine, and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

Miniprimer PCR (page does not exist) Miniprimer PCR uses a thermostable polymerase (S-Tbr) that can extend from short primers (smalligos) as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene.  Multiplex Ligation-dependent Probe Amplification Multiplex Ligation-dependent Probe Amplification (MLPA) permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR (see below).

Multiplex-PCR Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce Amplicon amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by Gel electrophoresis gel electrophoresis.

Nested PCR Nested PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3 of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.
Overlap-extension PCR Overlap-extension PCR a Genetic engineering genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.

Q-PCR Quantitative PCR (Q-PCR) used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision. QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or  Fluorophore fluorophore-containing DNA probes, such as TaqMan TaqMan, to measure the amount of amplified product in real time. It is also sometimes abbreviated to Real-time PCR RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to  RT-PCR reverse transcription PCR (see below), often used in conjunction with Q-PCR.

Reverse Transcription PCR (RT-PCR RT-PCR) for amplifying DNA from RNA.  Reverse transcriptase Reverse transcriptase reverse transcribes  RNA RNA into  CDNA cDNA, which is then amplified by PCR. RT-PCR is widely used in  Expression profiling expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of  Exons exons and  Introns introns in the gene. The 5 end of a gene (corresponding to the transcription start site) is typically identified by  RACE (biology) RACE-PCR (Rapid Amplification of cDNA Ends).

Solid Phase PCR (page does not exist) Solid Phase PCR encompasses multiple meanings, including Polony Polony Amplification (where PCR colonies are derived in a gel matrix, for example), Bridge PCR (primers are covalently linked to a solid-support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal step to favour solid support priming).

Thermal asymmetric interlaced PCR TAIL-PCR (page does not exist) TAIL-PCR) for isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures a degenerate primer is used to amplify in the other direction from the unknown sequence.

(Step-down PCR) a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles. PAN-AC (page does not exist) PAN-AC uses isothermal conditions for amplification, and may be used in living cells. Universal Fast Walking (page does not exist) Universal Fast Walking for genome walking and genetic fingerprinting using a more specific two-sided PCR than conventional one-sided approaches (using only one gene-specific primer and one general primer - which can lead to artefactual noise) by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends).

b. Model Systemsi. What is a good model See Wikipedia
Even with increasing number of researchers and facilities it may jus t not be possible to study even one organism in fullest depth. A model system id required for a situation when there are two basic questions of enquiry  the one best method for control and or prevention of any one human disease the other being does the present information provide every kind of information required to understand all its life mechanisms    

Concerning human biology and pathology in general there is always the issue which organism can provide the nearly closest answers to any of the human problems. Even assuming there could be one hypothetical organism whose biology is known and understood to the fullest extent   maximum there is no guarantee that this organisms could be used to understand and develop mechanisms for control and prevention of a problem in any other organism including human beings.

Yet the need for an organism is warranted which can be extensively studied intensively and extensively. In the course of accumulating information one after the other, many organisms have been used for unraveling a mechanism or development of testable theory based experiments and so on. For example whatever is known today about the genetics and molecular genetics is based a number of organisms.

For example Mendel performed his experiments with pea plants and discovered the famous two foundation laws of inheritance. In the paper read at ..  out of the total of 15,226 word, he devoted 706 words for a section  under the heading SELECTION OF THE EXPERIMENTAL PLANTS. In this section Mendel reveals his sense of accuracy in describing how and why he chose the pea plants. He wrote The value and utility of any experiment are determined by the fitness of the material to the purpose for which it is used, and thus in the case before us it cannot be immaterial what plants are subjected to experiment and in what manner such experiment is conducted. The experimental plants must necessarily

1. Possess constant differentiating characteristics. 2. .. In all, thirtyfour more or less distinct varieties of Peas were obtained from several seedsmen and subjected to a two years trial. Later he justified how the plants chosen were indeed fitting into his original enquiries on the regularity of the recurrence of the characters of the hybrid forms and their consistent proportions.

In the course of development of biology many organisms were chosen based on the need and basis of simple enquiry. In the process many organisms got selected more frequently because more and more of information got collected. This became the norm for the frequent choice of an organism for an experiment. But more recently the trend has changed so drastically that to dissect out an answer to a problem in one organism another related organisms has to be chosen. This is on the assumption that the solutions or leads obtained from the related organism can be extrapolated and extended to the test organism itself. For example any human related problem another mamma such as mouse or a monkey was the popular choice.    

After all these, now a clear concept on what a model organism should be, has emerged. Essentially a model organism has to be a non-human organism which has been extensively studied  for understanding a biological process in anticipation  that any findings with the model organisms would become applicable to other organisms. The situation has greater relevance to understanding human diseases. The model organism for investigation on causes and treatments for human disease so that the the findings may get applied to human situations. The need for a model organisms in such situations is also imperative on ethical grounds. This logic is now based on the consensus that all the organisms have a common ancestry. This is also due to the understanding that there has been evolutionary conservation in the genetic material and therefore the conservation of metabolic and developmental pathways. The only precaution that needs to be taken care of is that generalizations and simple extrapolations should not be generalized.  
Some Basic Quantifiable Criteria Needed in Model System
The organism to used should be easy to rear andor culture.
Its rearingoperation size should be convenient
It is economic and inexpensive to operate.
The organism for use should have a short life cycle

The organism must have an easy reproductive mechanism with very high rate of multiplication  
The organism to be used should have the ability to be grown in culture on well defined  simple well established media for genetic manipulations

It should have fitness into the experimental plan for anticipated results to have high potential for  providing economically important results in consequent experiments
The organism should have a wide audience of interested researchers and public as well.

There must be high level of positive interactions between and among these criteria.

Choice of a Model Organism
Generally the choice agree with most of the above parameters but may not agree with all. Over and above the above factors, the choice of an organism is made or becomes very stringent due to many reasons. The organism chosen based on the above parameters may satisfy all the above parameters but is not suitable because of lack of parallel comparisons with the test organism. The choices may have to be made on other considerations such as emphasis on using genetic models such as fruit fly or nematode (both with very rich genetic information, and high rates of multiplication in short life cycles), experimental models and or genomic models because of their key positions in the evolutionary tree. Historically and as mentioned earlier there are just a few including the NIH model organisms (NIH Model Organisms) which are characterized extensively genetically and in the sense of molecular biology.        

Choices of organism also include the negative traits that the organism may have. One organism may agree with all parameters but may have some technical limitations and difficulties such as large amounts of junk DNA. Organism for potential good use may have to be disregarded on other considerations such as ethical andor environmental considerations.

On the whole the many model organisms that have been developed and used may belong t any of the major life forms, from viruses to prokaryotes and eukaryotes including protists, fungi, plants, invertebrates and vertebrates.        

Organisms may have to be based on specific research objectives which makes the choice more difficult. They are Sexual selection and sexual conflict,  Hybrid zones, and ecological genomics, and ecological zones

Neurosota crassa
E coli


Summary of the article
The research article entitled, Effects of soil salt levels on the growth and water use efficiency of Atriplex canescens (Chenopodiaceae) varieties in drying soil, authored by Glenn and Brown (1998) described the performance of three plant varieties to varying degrees of salt and water concentrations.  It has long been established that maximal growth and development of any plant species are mainly dependent on optimal environmental conditions.  These include an ample supply of water, as well as nutrients from the soil, sun and sunlight.  Any significant decrease or absence of any of these factors may induce stress to a plant species.  Moreover, the combinatorial effect of at least two external factors may pose more harmful effects on a plant.

The quality of soil that is used for planting may also affect the growth and development of plants It should be understood that the soil serves as a water reservoir to the growing plant.  Water also serves as a matrix for transporting organic, as well as inorganic solutes to the plant.  The main region of entry for these solutes is the root of the plant, which is strategically located adjacent to the soil.  It has been suggested that the any perturbation of two external factors may impose an additive effect on the performance of a plant.  However, there are certain research reports that describe a more complicated mechanism, wherein the soil would mitigate conditions of water stress.  For example, soil that contains some amount of salt may assist in a plants survival when there is not enough water on the ground.  It is possible that plants may have adapted a response to water insufficiency by imbibing salts into its system, as this condition would retain moisture within the plant.

A number of water retention mechanisms have been earlier described, including the inherent increase in the number of salt ions within the leaves of the plant.  Another mechanism associated with water retention is the reduction in the frequency of opening the stomata of the leaf, thus reducing the transpiration of water to the environment.  It has also been suggested that plants may have learned to survive through scarce water conditions, by adjusting their osmotic potential to only require a minute amount of hydration each day.

The study of Glenn and Brown (1998) thus investigated the response of the C4 xerohalophytic shrub Atriplex canescens on salt stress.  This shrub is generally found in saltbushes in regions of North America, where the soil is often saline and dry.  Currently, there are six varieties of Atriplex and each could be differentiated through its degree of salt tolerance and water efficiency.  Despite the established features of this plant species, the investigators wanted to determine whether there was any interrelationship between the capacity of this species to adapt to salt and water stress.  The employment of three varieties of Atriplex would also allow the investigators to probe the actual association of salt tolerance to water stress.

Briefly, seedlings of Atriplex were transplanted to three different salinity conditions, namely 0, 85 and 520 molm3, representing suboptimal, optimal and supraoptimal conditions for plant growth.  The soil used in the study consisted of a mixture of river sand and peat-based potting soil, of which the mean initial water content was estimated to be 0.899 Lpot.  The seedlings were maintained in a greenhouse that had day temperatures ranging from 25oC to 35oC and light transmission of 65.  The seedlings were allowed to grow until 80 of the plant was wilted.  The plants were also measured for growth, as well as cation content and efficiency of use of water.  The results of the study showed that combinatorial effect of salt and water stress was not additive or increased, but instead, facilitated in the growth and survival of the plant.  The researchers found that seedlings that were subjected to higher salt conditions performed better under insufficient water conditions, as compared to seedlings that were subjected to optimal and suboptimal salt conditions.

Critique of the article
The research article of Glenn and Brown (1998) is a very informative report on the response of three varieties of Atriplex to varying external conditions.  The results of their study were novel because contrary to what has been upheld for decades, the plants they studied did not experience an additive impact when two external factors were introduced.  It has long been thought that the occurrence of two stressful factors to a plant would result in a double burden on the plant and that its capacity for growth and development would be significantly retarded.  However, this research report changes the belief that the effect of two stress factors would add up and impose developmental problems to plants.

The presentation of the rationale of the study was very clear.  The investigators explained the current concepts that have been established with regards to optimal growth and development of plants.  The citations of prior research reports are also comprehensive, as the investigators attempted to fully explain the basis and objectives of their study.  The methodology of the study was also good, wherein they attempted to keep the conditions of the experiment constant, except for the variable factors.  For example, the amount and components of the soil mixture was precisely determined based on weight.  In addition, the growth stage of the seedlings at the start of the study was also determined to be almost within the same range.

The data collected from the study was clearly presented, as seen in the graphs and tables.  The graphs for each plant variety clearly showed that there was indeed a difference in the growth and development of the seedlings based on the amount of salt in the soil.  The tables also showed that the investigators were cautious in the analysis of their data, as shown in their employment of the statistical test of analysis of variance (ANOVA).  This statistical test has the capability of identifying any significance differences between experimental conditions.  The investigators also employed the calculation of standard errors, which could also determine the robustness of the values of their measurements.  The data presented in the articles strongly support the conclusions of the investigation, wherein seedlings subjected to higher salt concentrations performed better than those subjected to normal or optimal external conditions.  The paper clearly presented their bottom line that salt and water stress did not impose an additive effect on the growth and development of the Atriplex varieties.

One weakness of the study was that the investigators did not attempt to determine any genetic factors that may have influenced such performance in salt conditions.  It is possible that there are certain genes that have been activated in the seedlings when a change in its external environment is detected.  For example, there are heat shock genes that are activated when an organism is exposed to extreme temperatures.  It is thus likely that the plants in this study were capable of activating salt and water shock genes that allowed them to adapt and ultimately survive such specific conditions.  The genetics aspect of this study may be performed through the rapid amplification of polymorphic DNA (RAPD) analysis, which can generate a DNA profile of each seedling.  It would also have been more informative if the investigators presented any tissues sections that would show how water and salt stress have affected the internal structure of the growing plants.
The tables and figures of the research article are clearly presented and easily understood.  The labels of each axis and column are properly labeled and thus as a reader, there is no difficulty in finding any information of interest from the report.  However, it would have been more interesting if the investigators provided images of the plant itself, possibly comparing a normal, affected and wilted plant.