Genetic Fingerprintng And DNA Profiling

Genetic fingerprinting, DNA testing, DNA typing, and DNA profiling are techniques used to distinguish between individuals of the same species using only samples of their DNA. Its invention by Sir Alec Jeffreys at the University of Leicester was announced in 1985. Two humans will have the vast majority of their DNA sequence in common. Genetic fingerprinting exploits highly variable repeating sequences called minisatellites. Two unrelated humans will be likely to have different numbers of minisatellites at a given locus. In STR profiling, which is distinct from DNA fingerprinting, PCR is used to obtain enough DNA to then detect the number of repeats at several loci. It is possible to establish a match that is extremely unlikely to have arisen by coincidence, except in the case of identical twins, who will have identical genetic profiles.
Genetic fingerprinting is used in forensic science, to match suspects to samples of blood, hair, saliva or semen. It has also led to several exonerations of formerly convicted suspects. It is also used in such applications as identifying human remains, paternity testing, matching organ donors, studying populations of wild animals, and establishing the province or composition of foods. It has also been used to generate hypotheses on the pattern of the human diaspora in prehistoric times.
Testing is subject to the legal code of the jurisdiction in which it is performed. Usually the testing is voluntary, but it can be made compulsory by such instruments as a search warrant or court order. Several jurisdictions have also begun to assemble databases containing DNA information of convicts.
The United Kingdom currently has the most extensive DNA database in the world, with well over 2 million records as of 2005: The National DNA Database (NDNAD). The size of this database, and its rate of growth, is giving concern to civil liberties groups in the UK, where police have wide-ranging powers to take samples and retain them even in the event of acquittal.
DNA fingerprinting methods
DNA fingerprinting begins by extracting DNA from the cells in a sample of blood, saliva, semen, or other appropriate fluid or tissue.
RFLP analysis
When DNA fingerprinting first began, restriction fragment length polymorphism (RFLP) analysis was used, though it has been almost completely replaced with newer techniques. RFLP analysis is performed by using a restriction enzyme to cut the DNA into fragments which are separated into bands during agarose gel electrophoresis. Next, the bands of DNA are transferred via a technique called Southern blotting from the agarose gel to a nylon membrane. This is treated with a radioactively-labeled DNA probe which binds to certain specific DNA sequences on the membrane. The excess DNA probe is then washed off. An X-ray film placed next to the nylon membrane detects the radioactive pattern. This film is then developed to make a visible pattern of bands called a DNA fingerprint. By using multiple probes targeting various polymorphisms in successive X-ray images, a fairly high degree of discrimination was possible. The primary drawback of RFLP is that the exact sizes of the bands are unknown and comparison to a molecular weight ladder is done in a purely qualitative manner. Many labs developed policies that described what they considered a unique band, but it was not standardized and led to DNA fingerprinting coming under harsh attack in People v. Castro 545 N.Y.S. 2d. 985 (Sup. Ct. 1989). RFLP was a very time consuming method which required relatively high quantity of good quality DNA to be used (such as a dime sized blood drop). This made typing degraded samples such as those from evidence that had been exposed to the elements fairly difficult.
PCR analysis
With the invention of the polymerase chain reaction (PCR), DNA fingerprinting took huge strides forward in both discriminating power and ability to recover information from very small starting samples. PCR involves the amplification of specific regions of DNA using a cycling of temperature and a thermostable polymerase enzyme along with sequence specific primers of DNA. Commercial kits that used single nucleotide polymorphisms (SNPs) for discrimination became available. These kits use PCR to amplify specific regions with known variations and hybridize them to probes anchored on cards, which results in a colored spot corresponding to the particular sequence variation.
One of the primary complaints against RFLP was that it was slow and required large quantities of DNA to be used. This led to the development of PCR-based methods which required smaller amounts of DNA that could also be more degraded than those used in RFLP analysis. Systems such as the HLA-DQ alpha reverse dot blot strips grew to be very popular due to their ease of use and the speed with which a result could be obtained, however they were not as discriminating as RFLP. It was also difficult to determine a DNA profile for mixed samples, such as a vaginal swab from a sexual assault victim.
AmpFLP
Another technique, AmpFLP, or amplified fragment length polymorphism was also put into practice during the early 1990's. This technique was also faster than RFLP analysis and used PCR to amplify DNA samples. It relied on variable number tandem repeat (VNTR) polymorphisms to distinguish various alleles, which were separated on a polyacrylamide gel using an allelic ladder (as opposed to a molecular weight ladder). Bands could be visualized by silver staining the gel. One popular locus for fingerprinting was the D1S80 locus. As with all PCR based methods, highly degraded DNA or very small amounts of DNA may cause allelic dropout (causing a mistake in thinking a heterozygote is a homozygote) or other stochastic effects. In addition, because the analysis is done on a gel, very high number repeats may bunch together at the top of the gel, making it difficult to resolve. AmpFLP analysis can be highly automated, and allows for easy creation of phylogenetic trees based on comparing individual samples of DNA. Due to its relatively low cost and ease of set-up and operation, AmpFLP remains popular in lower income countries.
STR analysis
The most prevalent method of DNA fingerprinting used today is based on PCR and uses short tandem repeats (STR). This method uses highly polymorphic regions that have short repeated sequences of DNA (the most common is 4 bases repeated, but there are other lengths in use, including 3 and 5 bases). Because different people have different numbers of repeat units, these regions of DNA can be used to discriminate between individuals. These STR loci (locations) are targeted with sequence-specific primers and are amplified using PCR. The DNA fragments that result are then separated and detected using electrophoresis. There are two common methods of separation and detection, capillary electrophoresis (CE) and gel electrophoresis.
The polymorphisms displayed at each STR region are by themselves very common, typically each polymorphism will be shared by around 5 - 20% of individuals. When looking at multiple loci, it is the unique combinations of these polymorphisms to an individual that makes this method discriminating as an identification tool. The more STR regions that are tested in an individual the more discriminating the test becomes.
From country to country different STR based DNA profiling systems are in use. In North America CODIS is prevalent, while in the UK the SGM+ system, which is compatible with The National DNA Database is in use. Whichever system is used, many of the STR regions under test are the same. These DNA profiling systems are based around multiplex reactions, whereby many STR regions will be under test at the same time.
Capillary electrophoresis works by electrokinetically (movement through the application of an electric field) injecting the DNA fragments into a thin glass tube (the capillary) filled with polymer. The DNA is pulled through the tube by the application of an electric field, separating the fragments such that the smaller fragments travel faster through the capillary. The fragments are then detected using fluorescent dyes that were attached to the primers used in PCR. This allows multiple fragments to be amplified and run simultaneously, something known as multiplexing. Sizes are assigned using labeled DNA size standards that are added to each sample, and the number of repeats are determined by comparing the size to an allelic ladder, a sample that contains all of the common possible repeat sizes. Although this method is expensive, larger capacity machines with higher throughput are being used to lower the cost/sample and reduce backlogs that exist in many government crime facilities.
Gel electrophoresis acts using similar principles as CE, but instead of using a capillary, a large polyacrylamide gel is used to separate the DNA fragments. An electric field is applied, as in CE, but instead of running all of the samples by a detector, the smallest fragments are run close to the bottom of the gel and the entire gel is scanned into a computer. This produces an image showing all of the bands corresponding to different repeat sizes and the allelic ladder. This approach does not require the use of size standards, since the allelic ladder is run alongside the samples and serves this purpose. Visualization can either be through the use of fluorescently tagged dyes in the primers or by silver staining the gel prior to scanning. Although it is cost effective and can be rather high throughput, silver staining kits for STRs are being discontinued. In addition, many labs are phasing out gels in favor of CE as the cost of machines becomes more manageable.
The true power of STRs is in its statistical power of discrimination. In the U.S.A., there are 13 loci (DNA locations) that are currently used for discrimination. Because these loci are independently assorted (having a certain number of repeats at one locus doesn't change the likelihood of having any number of repeats at any other locus), the power rule of statistics can be applied. This means that if someone has the DNA type of ABC, where the three loci were independent, we can say that the probability of having that DNA type is the probability of having type A times the probability of having type B times the probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion (1 with 18 zeros after it) or more.
Y-chromosome analysis
Recent innovations have included the creation of primers targeting polymorphic regions on the Y-chromosome (Y-STR), which allows resolution of multiple male profiles, or cases in which a differential extraction is not possible. Y-chromosomes are paternally inherited, so Y-STR analysis can help in the identification of paternally related males. Y-STR analysis was performed in the Sally Hemings controversy to determine if Thomas Jefferson had sired a son with one of his slaves.
Mitochondrial analysis
For highly degraded samples, it is sometimes impossible to get a complete profile of the 13 CODIS STRs. In these situations, mitochondrial DNA (mtDNA) is sometimes typed due to there being many copies of mtDNA in a cell, while there may only be 1-2 copies of the nuclear DNA. Forensic scientists amplify the HV1 and HV2 regions of the mtDNA, then sequence each region and compare single nucleotide differences to a reference. Because mtDNA is maternally inherited, directly linked maternal relatives can be used as match references, such as one's maternal grandmother's sister's son. A difference of two or more nucleotides is generally considered to be an exclusion. Heteroplasmy and poly-C differences may throw off straight sequence comparisons, so some expertise on the part of the analyst is required. mtDNA is useful in determining unclear identities, such as those of missing persons when a maternally linked relative can be found. mtDNA testing was used in determining that Anna Anderson was not the Russian princess she had claimed to be, Anastasia Romanov.
mtDNA can be obtained from such material as hair shafts and old bones/teeth.
Considerations when evaluating DNA evidence
In the early days of the use of genetic fingerprinting as criminal evidence, juries were often swayed by spurious statistical arguments by defense lawyers along these lines: given a match that had a 1 in 5 million probability of occurring by chance, the lawyer would argue that this meant that in a country of say 60 million people there were 12 people who would also match the profile. This was then translated to a 1 in 12 chance of the suspect being the guilty one. This argument is not sound unless the suspect was drawn at random from the population of the country. In fact, a jury should consider how likely it is that an individual matching the genetic profile would also have been a suspect in the case for other reasons. Another spurious statistical argument is based on the false assumption that a 1 in 5 million probability of a match automatically translates into a 1 in 5 million probability of innocence and is known as the prosecutor's fallacy.
When using RFLP, the theoretical risk of a coincidental match is 1 in 100 billion (100,000,000,000). However, the rate of laboratory error is almost certainly higher than this, and often actual laboratory procedures do not reflect the theory under which the coincidence probabilities were computed. For example, the coincidence probabilities may be calculated based on the probabilities that markers in two samples have bands in precisely the same location, but a laboratory worker may conclude that similar -- but not precisely identical -- band patterns result from identical genetic samples with some imperfection in the agarose gel. However, in this case, the laboratory worker increases the coincidence risk by expanding the criteria for declaring a match. Recent studies have quoted relatively high error rates which may be cause for concern [1]. Because of this, arbitrary low ceilings were put on match probabilities used in RFLP analysis rather than the higher theoretically computed ones [2]. Today, RFLP has become widely disused due to these difficulties in interpretation.
STRs do not suffer from such subjectivity and provide much better powers of discrimination, for unrelated individuals (of the order of 1 in 10^29 if using a full profile) It should be noted that figures of this magnitude are not considered to be statistically suportable by scientists in the UK, for unrelated individuals with full matching DNA profiles a match probability of 1 in a billion (one thousand million) is considered statistically supportable (Since 1998 the DNA profiling system supported by The National DNA Database in the UK is the SGM+ DNA profiling system which includes 10 STR regions and a sex indicating test, this test updated the SGM DNA profiling system on which the National DNA Database was founded in 1995. The SGM system included 6 out of the 10 STR regions used in the SGM+ system and the same sex indicating test, however the discriminating power of the SGM system was only considered to be supportable at 1 in a million) . However, with any DNA technique, the cautious juror should not convict on genetic fingerprint evidence alone if other factors raise doubt. Contamination with other evidence (secondary transfer) is a key source of incorrect DNA profiles and raising doubts as to whether a sample has been adulterated is a favorite defense technique. More rarely, Chimerism is one such instance where the lack of a genetic match may unfairly exclude a suspect.
When evaluating a DNA match, the following questions should be asked:
· Could it be an accidental random match?
· If not, could the DNA sample have been planted?
· If not, did the accused leave the DNA sample at the exact time of the crime?
· If yes, does that mean that the accused is guilty of the crime?
Fake DNA evidence
The value of DNA evidence has to be seen in light of recent cases where criminals planted fake DNA samples at crime scenes. In one case, a criminal even planted fake DNA evidence in his own body: Dr. John Schneeberger of Canada raped one of his sedated patients in 1992 and left semen on her underwear. Police drew Schneeberger's blood and compared its DNA against the crime scene semen DNA on three occasions, never showing a match. It turned out that he had surgically inserted a Penrose drain into his arm and filled it with foreign blood and anticoagulants.
Cases
In the 1920s, Anna Anderson claimed that she was Princess Anastasia Romanov of Russia; in the 1980s her cremated remains were tested and seemed to show that she was no relation to the Romanovs.
In 1987, British baker Colin Pitchfork was the first criminal caught using DNA fingerprinting in Leicester, the city where it was first discovered.
In 1987, Florida rapist Tommie Lee Andrews was the first person in the United States to be convicted as a result of DNA evidence, for raping a woman during a burglary; he was convicted on 6 November 1987 and sentenced to 22 years in prison. [3] [4]
In 1989, Chicago man Gary Dotson was the first person whose conviction was overturned using DNA evidence.
In 1991, Allan Legere was the first Canadian to be convicted as a result of DNA evidence, for four murders he had committed while an escaped prisoner in 1989. During his trial, his defense argued that the relatively shallow gene pool of the region could lead to false positives.
In 1992, DNA evidence was used to prove that Nazi doctor Josef Mengele was buried in Brazil under the name Wolfgang Gerhard.
In 1993, Kirk Bloodsworth was the first person to have been convicted of murder and sentenced to death, whose conviction was overturned using DNA evidence.
The science was made famous in the United States in 1994 when prosecutors heavily relied on — and through expert witnesses exhaustively presented and explained — DNA evidence allegedly linking O.J. Simpson to a double murder. The case also brought to light the laboratory difficulties and handling procedure mishaps which can cause such evidence to be significantly doubted.
In 1994, RCMP detectives successfully tested hairs from a cat known as Snowball, and used the test to link a man to the murder of his wife, thus marking for the first time in forensic history the use of non-human DNA to identify a criminal.
In 1998, Dr. Richard J. Schmidt was convicted of attempted second-degree murder when it was shown that there was a link between the viral DNA of the human immunodeficiency virus (HIV) he had been accused of injecting in his girlfriend and viral DNA from one of his patients with full-blown AIDS. This was the first time viral DNA fingerprinting had been used as evidence in a criminal trial.
In 2002, DNA testing was used to exonerate Douglas Echols, a man who was wrongfully convicted in a 1986 rape case. Echols was the 114th person to be exonerated through post-conviction DNA testing.
In 2003, Welshman Jeffrey Gafoor was convicted of the 1988 murder of Lynette White, when crime scene evidence collected 12 years earlier was re-examined using STR techniques, resulting in a match with his nephew.[5] This may be the first known example of the DNA of an innocent yet related individual being used to identify the actual criminal, via "familial searching".
In June of 2003, because of new DNA evidence, Dennis Halstead, John Kogut and John Restivo won a re-trial on their murder conviction. The three men had already served eighteen years of their thirty plus year sentences.
The trial of Robert Pickton is notable in that DNA evidence is being used primarily to identify the victims, and in many cases to prove their existence.
In March 2003, Josiah Sutton was released from prison after serving four years of a twelve year sentence for a sexual assault charge. Questionable DNA samples taken from Sutton were retested in the wake of the Houston Police Department's crime lab scandal of mishandling DNA evidence.
In December 2005, Robert Clark was proven innocent of a 1981 attack on an Atlanta woman after serving twenty four years in prison. Mr Clark is the 164th person in United States and the fifth in Georgia to be freed using post-conviction DNA testing

Modified Bringal(GM Bringal) on your Dining Table

Modified brinjal on your table, good or bad?
New Delhi: The Genetic Engineering Approval Committee (GEAC), the biotechnology regulator, on Wednesday approved the commercialisation of genetically-modified Bt brinjal.
Bt brinjal still needs the government's nod before its release in the market. If it gets the nod, Bt brinjal will be the first genetically-modified food in India.
Members of the committee, which met here, said the genetically-modified crop had the potential to increase yields by a significant extent.
But opposing the GEAC decision, farmers' union the All India Kisan Sabha said: "There are many unresolved issues surrounding the environmental release of the transgenic vegetable as well as genuine concerns expressed over its safety for human consumption. There is also the added threat of all future seeds and therefore Indian agriculture coming under the control of global MNCs and the charging of extortionate prices from Indian farmers."
The introduction of the genetically-modified brinjal is part of an USAID programme called Agri-Biotechnology Support Programme (ABSP) under which the Indian Institute of Vegetable Research, Varanasi; University of Agricultural Sciences, Dharwad and Tamil Nadu Agricultural University, Coimbatore are working with Monsanto and Mahyco.
In a statement, the Sabha said: "It has been pointed out that some of the 'experts' in the GEAC have conflicts of interest. Certain experts on the committee are reported to have expressed strong objections which were however not taken into account.
"If the GEAC carries forward the environmental release of Bt brinjal floodgates will be opened for nearly 60 genetically-modified food crops in India, some of which are already in the pipeline like rice, corn, okra etc."
It pointed out that the European Union has banned genetically-modified food crops.
Demanding that more studies be carried out before Bt brinjal is introduced and that the decision making process of the GEAC be more transparent, Sabha president S. Ramachandran Pillai and general secretary K. Varadha Rajan said in their joint statement: "Concerns regarding the health and environmental risks associated with GM crops are too serious to be disregarded. The seed monopolies that threaten Indian agriculture and farmers' livelihoods should also be reined in."
Greenpeace spreading misinformation on Bt brinjal: Ramesh
New Delhi: Minister of State for Environment and Forests Jairam Ramesh Wednesday slammed Greenpeace, a voluntary organisation, for spreading "wrong information" about the commercialisation of genetically modified Bt brinjal.
Opposing the introduction of Bt brinjal in India, Greenpeace has said India is the centre of origin and diversity of brinjal and if Bt brinjal is approved, this would be the first time in the world that a genetically modified crop is allowed in its centre of origin/diversity, risking bio-diversity.
"They have spread wrong information. I condemn it. I am for transparency. There is a way to conduct discourse," Ramesh told reporters here.
He said the government would hear the views of those favouring and opposing the introduction of Bt brinjal before taking a final decision.
"There are arguments in favour of introduction. There are arguments against introduction," he said.
Ramesh's comments came hours after the Genetic Engineering Approval Committee (GEAC), the biotechnology regulator, approved the commercialisation of genetically modified Bt brinjal.
If it gets the nod, Bt brinjal would be the first genetically modified food in India.
Source: Indo-Asian News Service

Why GM foods are Hamrful...An Interviews with Susheel Dwivedi

you have spoken at various platforms all over the India against genetically modified crops. Are they harmful to human beings, animals and other plants?
There is evidence that they are harmful. GMOs (genetically modified organisms), first of all, do not bring any benefit to either health or the economy in terms of increasing production.
Those who support use of GMO say they increase production, but they don't say that it is only because pesticides are attached to them. They kill insects which normally share some of nature's bounty.
You create seeds that are genetically dependent on some kind of pesticide and that pesticide will kill any kind of life.
For example, if you have a herbicide like Roundup, and that Roundup is owned by Monsanto, it will kill absolutely anything that is green in sight; grass, trees, plants, anything that is green and in soil. Except the crop that I want to grow, everything else will be killed.
What they do not want to tell is what has been destroyed and left in the plant. When you kill plants, on the plant there are insects, worms and nitrogen fixing bacteria; all that gets destroyed. This is happening year after year. If one herbicide stops working, they come up with another one. So, it is bad for the soil, other plants and those who consume it.
It was reported that these pesticide-herbicide injected seeds affect the nearby farms where there are no GMOs...
Yes, wind will blow seeds from one farm to another. Then, Monsanto will send their inspectors who threaten the farmers for growing their seeds when the seeds reached the farm having been blown by wind.
Then, Monsanto will say, either you pay me for using our seeds or I will take you to court. There are several such cases reported in the United States and Canada where they have taken farmers to court and ruined their lives.
Is it because these multinational companies like Monsanto are so powerful that we do not hear many scientists or politicians talking about the harmful effects of GMOs?
Absolutely! This is why I call my latest book Corrupt To The Core. It is not just Monsanto owned by Pfizer; it is only that it is the company that has the nastiest name, but there are other companies doing the same thing.
If it is harmful to flora and fauna, is it not harmful to human beings who consume these crops?
Of course, it is! But nobody asks that question. Do you think the scientists do not know this? It is because the whole thing has become so corrupt in the hands of these companies.
In my book, I quote from a speech given in Canada in 1990 by a Monsanto executive. He said, 'MNCs will soon rule the world through intellectual property rights (IPR). We can take over the White House, the Parliament of England, France, Japan and Germany, and once we do that, we will see that China and India would come along and then we can take Africa for granted.'
I quoted this from a published paper in 1990. This is precisely what has happened.
Governments are being taken over by multinationals and governments are no longer listening to people and not working in public interest.
Is India the new attraction because a majority of the population is involved in farming?
First of all, America is not run by the American people any more; it is being run by these multinational corporations.
America and the other G-8 countries are on the verge of becoming bankrupt. America is a saturated economy, owes so much money to the world and is still burning a billion dollars a day on war and there is no hope of any recovery. Naturally, their next target will be countries like India.
Why did they choose Bt brinjal to market first in India: a simple vegetable that many people do not even like?
Exactly, that is the question. Many people don't eat brinjal, children hate it. Still, why do you think brinjal was chosen to be the first Bt food crop?
This is a very calculated move on their part.
Brinjal is an unimportant vegetable but it belongs to the Solanaceae family, that also has popular vegetables like tomatoes, potatoes and chillies. The Bt gene is capable of spreading to other crops from the same family when it is being blown in the wind. This way, they contaminate the entire food and vegetable supply.
Agriculture Minister Sharad Pawar and the others have already started talking about Bt sweetcorn and Bt sugarcane. Although Environment Minister Jairam Ramesh has rejected Bt brinjal for now, his colleagues are after his skin.
The prime minister has, for the first time, ordered to look at whether Bt cotton was good or bad. Why do you do it after all the fiasco? It should have been done earlier.
Why is it that the scientific community is not raising its voice against GM seeds?
There is corruption everywhere. It is no different in the US or Canada. Their jobs depend on this. Sadly, neither the media nor the scientific journals are reporting the facts.
Your name is synonymous with food safety. Is there food safety in any of the countries in the world?
Cuba will take the lead; their food is completely organic. India is a mixed bag. China is very corrupt, whether it is food or milk.
You said not only GMOs, but hormones, antibiotics, pesticides and slaughter house wastes also have to be banned. . .
Yes. The latest is the GMOs. For the last fifty years none of these products has been proven to be safe. It is the responsibility of the company to prove that they are safe because they are going to make money.
We now know that with the use of all these, not only cancer but other chronic diseases like reproductive disorders, neurological disorders, diabetes, and other new diseases have reached epidemic proportions.
Because of this, the European Union, led by Denmark, has banned three of these products (hormones, antibiotics and slaughter house wastes) entering the food supply.
But the US and Canada say they are alright. Yet, no country wants to buy Canadian beef. Australia and New Zealand now export food materials free of these five substances to the EU.
You said at a function in India that the release of Bt brinjal will be the beginning of the end of Indian agriculture. That's a scary statement. . .
But that is the truth. If you allow Bt brinjal to enter the farms in India, everything will automatically becomes Bt. Once Bt brinjal is widely grown, they do not have to introduce Bt potato, Bt chilly and Bt tomato; the Bt gene will automatically get into the food supply.
In no time, potato, tomato, chilli will contain the Bt gene and there will be no ordinary tomato or potato crop that can be grown by the farmer. Only the Bt species will thrive. Even crops like rice and maize can get contaminated.
With only Bt seeds in the farms, all the seeds needed for Indian agriculture will be owned by a private company and your thousands of varieties of, say, brinjal will automatically get wiped out. That is because you will be forced to buy only one kind of seed.
That will be the beginning of the end of Indian agriculture. Remember they are not in India to help India fight hunger; they are here to make money.
How frightening is the situation for Nature and human beings?
Very frightening! The main reason America is going bankrupt is the huge expense on healthcare. They spend 16 per cent of their GDP on healthcare. If the new bill passes, it will become 20 per cent. Americans do not live any longer than other people in the world.
The fundamental question everyone asks is, what does every living being live for? Every living being lives only for food, the rest are all luxuries. We have started depleting our agricultural sector. From 80 per cent of the Indian economy, it has become 60 per cent. Now their plan is to make it 50 per cent in ten years.
The government thinks it is a good idea as there will be more progress in the cities. But who will feed all these people?

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How to detect Food Adulteration

How To Detect Food Adulterants

By Susheel Dwivedi
mobile-09435946180,
www.bioguuindia.blogspot.com
Adulterants, both harmful and simple, can be detected easily through small tests. These tests can be done at home too. What one needs is a set of equipment and chemicals and the culprits can be found out easily through these simple anti-adulteration tests.
Here are a few such tests as suggested by the Union Ministry of Health and Family Welfare, Government of India:

Vegetable oils:

Vegetable oils are generally mixed with castrol oil or argemone oil to make quick profits. These adulterants can be detected by the following two tests: In case of castrol oil: Take 1 ml. of vegetable oil in a clean dry test tube. Add 10 ml. of acidified petroleum ether. Shake vigorously for 2 minutes. Add 1 drop of ammonium molybdate reagent. The formation of turbidity indicates presence of castor oil in the sample.
In case of argemone oil: Add 5 ml concentrated HNO3 to 5 ml.sample. Shake carefully. Allow to separate. Yellow, orange yellow or crimson colour in the lower acid layer indicates adulteration.

Ghee:

Ghee is generally mixed with mashed potato or sweet potato to make it weighty and creamy. Often vanaspati is also added to Ghee.
In case of mashed potato or sweet potato: Boil 5 ml. o the sample in a test tube. Cool and put a drop of iodine solution. Blue colour indicates presence of starch. Colour disappears on boiling and reappears on cooling.
In case of vanaspati: Take 5 ml. of the sample in a test tube. Add 5 ml. of hydrochloric acid and 0.4 ml of 2 per cent furfural solution or sugar crystals. Insert the glass stopper and shake for 2 minutes. Development of a pink or red colour indicates presence of vanaspati in Ghee.
Often old ghee (rancid stuff) is added. To detect this, take one teaspoon of melted sample and 5 ml. of HCL in a stoppered glass tube. Shake vigorously for 30 seconds. Add 5 ml. of 0.1 per cent of ether solution of phloroglucinol. Restopper and shake for 30 seconds and allow to stand for 10 minutes. A pink or red colour in the lower acid layer indicates rancidity.

Synthetic colours in food items:

To find out whether synthetic colouring matter is used in food items, pour 2 gms. of filtered fat dissolved in ether. Divide into 2 portions. Add 1 ml. of HCL to one tube. Add 1 ml. of 10 per cent NaOH to the other tube. Shake well and allow to stand. Presence of pink colour in acidic solution or yellow colour in alkaline solution indicates added colouring matter.

Honey:

Honey is good for health and it has several curative properties. But honey is generally adulterated with invert sugar or jaggery. There are two tests to find out whether the honey in question is pure or adulterated.
Fiehe’s Test: Add 5 ml. of solvent ether to 5 ml. of honey. Shake well and decant the ether layer in a petri dish. Evaporate completely by blowing the ether layer. Add 2 to 3 ml. of resorcinol (1 gm. of resorcinol resublimed in 5 ml. of concentrated HCL.). Appearance of cherry red colour indicates presence of sugar/jaggery.
Aniline chloride Test: Take 5 ml. of honey in a porcelain dish. Add aniline chloride solution (3 ml of aniline and 7 ml. of 1:3 HCL) and stir well. Orange red colour indicates presence of sugar.

Pulses and Besan:

Besan atta or pulses are adulterated with Kesari dal (Lathyrus sativus). To find out the adulterant, add 50 ml. of diluted HCL to a small quantity of dal and keep on simmering water for about 15 minutes. The pink colour, if developed, indicates the presence of Kesari dal.
Pulses are also adulterated with metanil yellow dye. To find this out, add concentrated HCL to a small quantity of dal in a little amount of water. Immediate development of pink colour indicates the presence of metanil yellow and similar colour dyes.
To find out whether lead chromate is used in the pulses, shake 5 gm. of pulses with 5ml. of water and add a few drops of HCL. Pink colour indicates lead chromate.

Wheat flour or atta:

Atta is generally contaminated with excessive sand and dirt. Shake a little quantity of sample with about 10 ml of carbon tetrachloride and allow to stand. Grit and sandy matter will collect at the bottom.
Often chalk powder is used in atta. To find out, shake sample with diluted HCL. Effervescence indicates chalk

Common spices:

Common spices like turmeric, chilly and curry powder are also adulterated by colours.
Extract the sample with petroleum ether and add 13N H2SO4 to the extract. Appearance of red colour (which persists even upon adding little distilled water) indicates the presence of added colours. However, if the colour disappears upon adding distilled water the sample is not adulterated.
Spices (ground) are adulterated by red bran and saw dust. Sprinkle on water surface. Powdered bran and sawdust float on the surface.
Coriander powder is adulterated with dung powder. To find out, soak in water. Dung will float and can be easily detected by its foul smell.

Chillies:

Brick powder, grit, sand, dirt, filth, etc are used in chillies, especially chilli powder. Pour the sample in a beaker containing a mixture of chloroform and carbon tetrachloride. Brick powder and grit will settle at the bottom.

Turmeric:

Lead chromate is used to give turmeric its natural color. Ash the sample. Dissolve it in 1:7 sulphuric acid (H2SO4) and filter. Add 1 or 2 drops of 0.1 per cent dipenylcarbazide. A pink colour indicates presence of lead chromate.

Cumin seeds:

Grass seeds coloured with charcoal dust is used. Rub the cumin seeds on palms. If palms turn black adulteration in indicated.

Asafoetida (Heeng):

Items like soap stone and other earthy matter is used for adulteration. Shake a little quantity of powdered sample with water. Soap stone or other earthy matter will settle at the bottom.
In case chalk is used as an adulterant, shake sample with carbon tetrachloride (CCl4). Asafoetida will settle down. Decant the top layer and add diluted HCL to the residue. Effervescence shows presence of chalk.

Foodgrains:

Foodgrains contain hidden insect infestations. To test the adulterants, take a filter paper impregnated with ninhydrin (1 per cent in alcohol). Put some grains on it and then fold the filter paper and crush the grains with hammer. Spots of bluish purple colour indicate presence of hidden insect infestation.

Important Tips FOR CSIR NET Life Science Exam 2010

1. Effect of EDTA on Bacterial cell is : 1. Effect of EDTA on Bacterial cell is Remove Mg++ ions digest polymeric compounds Removes lipid molecules removes insolubles cell debris. EDTA (ethylenediaminetetraacetic acid). The EDTA molecule can bind to metal ions by forming six bonds to it - two from nitrogen atoms in amino groups and four from oxygen atoms in carboxyl groups. Ca and Mg ions form the salt bridges in gram-negative bacteria binding polysaccharides on the surface of the cell wall. They also chelate the divalent ions of the lipoprotein and lipopolysaccharides of the cell wall. Antimicrobials in Food By P. Michael Davidson, John Nikolaos Sofos, Alfred Larry Branen

2. Klenow fragment can : 2. Klenow fragment can only polymerise DNA on a single strand only degrade DNA both none Uses of Klenow fragment Synthesis of double-stranded DNA from single-stranded templates. Filling in recessed 3' ends of DNA fragments. Digesting away protruding 3' overhangs. Preparation of radioactive DNA probes. In some situations, the 3' -> 5' exonuclease activity of Klenow fragment is either undesirable or not necessary. By introducing mutations in the gene that encodes Klenow, forms of the enzyme can be expressed that retain polymerase activity, but lack any exonuclease activity. These forms are the enzyme are usually called exo- Klenow fragment.

3. which one of the following acts as inducer of lac operon : 3. which one of the following acts as inducer of lac operon lactose allolactose permease ß galactosidase The tetrameric lac repressor protein, has four binding sites for allolactose. When those sites become occupied, the protein undergoes a change in shape (allosteric modification), which causes it to dissociate from the operator site, allowing transcription to proceed. Thus allolactose induces the lac operon by a process of de-repression. In order for lactose to induce the operon, there must already be present a low level of permease to get the lactose into the cell and a low level of ß-galactosidase to convert the lactose to allolactose. Mutants that totally lack either the permease or ß-galactosidase cannot be induced by lactose.

4. Most base substitution in -10 to -35 base pair region causes : 4. Most base substitution in -10 to -35 base pair region causes increase in promoter function decrease promoter function does not affect promoter function increase operator function A specific region just upstream from a gene that acts as a binding site for transcription factors and RNA polymerase during the initiation of transcription. In prokaryotes, the promoter consists of two short sequences at -10 and -35 positions upstream from the transcription start site. The sequence at -10 is called the Pribnow box, or the -10 element, and usually consists of the six nucleotides TATAAT. The Pribnow box is absolutely essential to start transcription in prokaryotes. The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows a very high transcription rate.

5. Linkers are : 5. Linkers are Double stranded DNA with blunt ends Single stranded DNA with sticky ends Single stranded DNA with blunt ends Single stranded DNA with sticky ends Sticky end and Blunt end are the two possible configurations resulting from the breaking of double-stranded DNA. DNA exhibits a stabilizing interaction between complementary base pairs, providing specificity to the pairing of two strands of DNA. If two complementary strands of DNA are of equal length, then they will terminate in a blunt end, as in the following example. (linkers) 5'-CpTpGpApTpCpGpCpTpApGpT-3' 3'-GpApCpTpApGpCpGpApTpCpA-5' However, if one strand extends beyond the complementary region, then the DNA is said to possess an overhang or it has a sticky end. (Adapters) 5'-ApTpCpTpGpApCpT-3‘ 3'-TpApGpApCpTpGpApCpTpApCpG-5' These linkers play a vital role in genetic Engineering. They are used in cloning Experiments.


6. Leucine Zipper has : Leucine Zipper has Leu residue at every 7th position of a -helix Leu residue at every 4th position of a -helix Leu residue at every 7th position of ß-sheet Leu residue at every 4th position of ß-sheet D represents Leu residue A structure, referred to as the 'leucine zipper’ explain how some eukaryotic gene regulatory proteins work. The leucine zipper consist of a periodic repetition of leucine residues at every seventh position. The segments containing these periodic arrays of leucine residues seem to exist in an alpha-helical conformation. The leucine side chains extending from one alpha-helix interact with those from a similar alpha helix of a second polypeptide, facilitating dimerization; the structure formed by cooperation of these two regions forms a coiled coil. These leucines form the hydrophobic core of a coiled coil.

7. Which one can be used to cut a methionine between two polypeptide : Which one can be used to cut a methionine between two polypeptide Factor Xa Thrombin Cyanogen bromide none Cyanogen bromide attacks on the carboxylate side of methionine, converting it to homoserine. The chemistry of the reaction involves the nucleophilic attack of the thioether sulphur on the carbon in CNBr, followed by the cyclization of iminolactone, which is hydrolysed in water resulting in peptide cleavage. Thrombin is a serine protease that possesses trypsin-like behavior in that it prefers to cleave its substrates after arginine residues. Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg. Chemical Reagents for Protein Modification By Roger L. Lundblad thioether

8. Thrombin cuts the polypeptide at : . Thrombin cuts the polypeptide at next to Gly next to Arg next to Met next to Lys The thrombin molecule contains two chains. The A chain is composed of 36 residues and is non-essential for proteolytic activities. The B chain is composed of 259 amino acids and is derived from the carboxyl terminal sequence of prothrombin. The B chain contains the three active site amino acids, His57, Asp102, and Ser195. Thrombin is a serine protease that possesses trypsin-like behavior in that it prefers to cleave its substrates after arginine residues.


9. Primer extension is used for identification of : Primer extension is used for identification of 3' end 5' end both none A primer is a strand of nucleic acid that serves as a starting point for DNA replication. They are required because the enzymes that catalyze replication, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3’end of the primer, and copies the opposite strand. In primer extension, a short antisense 5' end-labeled DNA primer (usually a synthetic oligonucleotide, but sometimes a small restriction fragment) is hybridized to RNA, usually total cellular RNA, then DNA is synthesized from this primer using reverse transcriptase. RTase will copy the RNA from the site of primer annealing to the 5'-end of the RNA molecule. The reactions are then analyzed by electrophoresis in sequencing gels in lanes adjacent to Sanger sequencing reactions of DNA containing the gene of question, using the same primer as that used in the PE analysis. The transcription initiation site (usually) can then easily be identified as the band in the sequencing reaction directly parallel to the run-off reverse transcript.

10. S1 nuclease analysis can be used for identification of : . S1 nuclease analysis can be used for identification of 3' end 5' end both none S1 nuclease is an endonuclease that is active against single-stranded DNA and RNA molecules. It is five times more active on DNA than RNA. S1 nuclease analysis Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe. At the end of the reaction period, nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by either autoradiography or Southern hybridization. The method can be used to quantify RNAs, to map the positions of introns and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates

11. Adapters has : 11. Adapters has one sticky end and one blunt end both sticky ends both blunt ends none Adapters are linkers with cohesive ends or a linker digested with RE, before ligation. The most widely used definition is cut linkers also called as adapters.They are not perfectly double stranded non single stranded. By adding adaptors to the ends of a DNA, sequences that are blunt can be converted into cohesive ends. Adaptor molecules are synthesized so that the blunt end is the same as ‘natural’ DNA, but the sticky end is different. The 3'-OH terminus of the sticky end is the same as usual, but the 5'-P terminus is modified; it lacks the phosphate group, and is in fact a 5'-OH terminus. DNA ligase is unable to form a phosphodiester bridge between 5'-OH and 3'-OH ends. The result is that, although base pairing is always occurring between the sticky ends of adaptor molecules, the association is never stabilized by ligation. Adaptors can therefore be ligated to a DNA molecule but not to themselves. After the adaptors have been attached, the abnormal 5'-OH terminus is converted to the natural 5'-P form by treatment with the enzyme polynucleotide kinase, producing a sticky-ended fragment that can be inserted into an appropriate vector. 5'-ApTpCpTpGpApCpT-3‘ 3'-TpApGpApCpTpGpApCpTpApCpG-5'

12. What is the ratio of absorbance of UV light by pure DNA at 260 nm and 280nm wavelength : 12. What is the ratio of absorbance of UV light by pure DNA at 260 nm and 280nm wavelength 1.8 4.8 10.2 7.8 Since nucleotides, RNA, ssDNA, and dsDNA all absorb at 260 nm, they will contribute to the total absorbance of the sample. Therefore, to ensure accurate results, of purity, ratio of absorbance of UV light by DNA at 260 nm and 280nm wavelength is calculated. A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. The resultant 260:280 ratio for the nucleic acid being studied will be approximately equal to the weighted average of the 260/280 ratios for the four nucleotides present. The generally accepted ratios of 1.8 and 2.0 for DNA and RNA respectively, are "rules of thumb". The actual ratio will depend on the composition of the nucleic acid. RNA will typically have a higher 260/280 ratio due to the higher ratio of Uracil compared to that of Thymine. Guanine: 1.15 Adenine: 4.50 Cytosine: 1.51 Uracil: 4.00 Thymine: 1.47 260/280 ratio

13. The most abundant enzyme in the biosphere : 13. The most abundant enzyme in the biosphere Amylase Rubisco DNA polymerase RNA polymerase Ribulose-1,5-bisphosphate carboxylase/oxygenase, most commonly known by the shorter name RuBisCO, is an enzyme that is used in the Calvin cycle to catalyze the first major step of carbon fixation, a process by which the atoms of atmospheric carbon dioxide are made available to organisms in the form of energy-rich molecules such as sucrose. RuBisCO catalyzes either the carboxylation or the oxygenation of ribulose-1,5-bisphosphate (also known as RuBP) with carbon dioxide or oxygen. RuBisCO is very important because it catalyzes the most commonly-used chemical reaction by which inorganic carbon enters the biosphere. RuBisCO is also the most abundant protein in leaves. It is found in plants, algae, cyanobacteria, phototropic and chemoautotropic proteobacteria .

14. Method used for differentiating real genes from chance ORF's is . Method used for differentiating real genes from chance ORF's is CpG island codon bias Homology search and transcript analysis All of above An open reading frame (ORF) is a portion of an organism's genome which contains a sequence of bases that could potentially encode a protein. In a gene, ORFs are located between the start-code sequence (initiation codon) and the stop-code sequence (termination codon). However, short ORFs can also occur by chance outside of genes. Usually ORFs outside genes are not very long and terminate after a few codons. CpG islands are genomic regions that contain a high frequency of CG nucleotides. CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates. DNA regions >500 bp with a GC content >55% and observed CpG/expected CpG of 0.65 were more likely to be the true CpG islands. In most genomes not all members of a codon family are used with equal frequency. Eg: Humans,use GTG four times more frequently than GTA for coding valine. If an ORF contains high frequency of rare codons then it is probably not a gene. Homology search : When a test sequence is BLASTed and if 30% of amino acids matches with the database, then it can be confirmed a real gene. Transcript analysis: For many model organisms the EST’s and cDNA sequences are used.if the sequence of ORF matches the sequence of transcript. Then the ORF is a real gene.

15. Hybrid of trp and lac promoter is called : Hybrid of trp and lac promoter is called lrp lap tac trc Tac is a strong promoter and is the hybrid of the trp and lac promoters, containing -35 region of trp fused with -10 region of lacUV5(a form where there are 2 mutations in -10 region, leading to enhanced expression) promoter.It is induced by Lactose and IPTG.It is regulated by lac repressors and is independent of cAMP regulation. Eg of vectors containing tac promoters are pkk223,pkk233 and others. Trc is also a hybrid promoter of trp and lac,containing -35 region of trp fused with -10 region of lacUV5.These 2 regions are separated by 17bp whereas in tac they are separated by 16bp. Lap is a latency associated prmoter and is not a hybrid promoter.

16. Which one is not used in making genetic maps : 16. Which one is not used in making genetic maps RFLP STR SNP EST's Genetic maps depict relative positions of loci based on the degree of recombination. This approach studies the inheritance/assortment of traits by genetic analysis. Physical maps show the actual (physical) distance between loci (in nucleotides). This approach applies techniques of molecular biology. Eg: Restriction mapping, EST mapping A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci, that can be observed. Eg: RFLP, STR, SNP, microsatellites etc An Expressed Sequence Tag is a tiny portion of an entire gene that can be used to help identify unknown genes and to map their positions within a genome. ESTs are small pieces of DNA sequence (usually 200 to 500 nucleotides long) that are generated by sequencing either one or both ends of an expressed gene. The idea is to sequence bits of DNA that represent genes expressed in certain cells, tissues, or organs from different organisms and use these "tags" to fish a gene out of a portion of chromosomal DNA by matching base pairs.

17. which is true for homeodomain : which is true for homeodomain was discovered in homeotic genes has DNA binding segment related to helix-turn-helix motif is enclosed in homeobox All of above Homeotic genes are defined by a DNA sequence known as the homeobox, which is a sequence of 180 nucleotides that code for a protein domain known as the homeodomain. The first genes found to encode homeodomain proteins were Drosophila developmental control genes, in particular homeotic genes, from which the name homeobox was derived. However, many homeobox genes are not homeotic genes; the homeobox is a sequence motif, while "homeotic" is a functional description for genes that cause homeotic transformations. The protein products of homeotic genes belong to a class of proteins known as transcription factors, all of which are capable of binding to DNA, thereby regulating the transcription of genes. The homeobox sequence codes for a 60 amino acid helix-turn-helix protein known as the homeodomain. The homeodomain acts as an "on/off" switch for gene transcription by binding to specific sequence enhancers of a gene, which either activates or represses the gene.

18. Plasmids are found in - . Plasmids are found in - bacteria yeast mouse both a and b Small, circular, extrachromosomal DNA molecules. They can replicate independently of the genome, and are found in numbers ranging from one per cell to hundreds per cell (this is called "copy number"). Plasmids frequently carry genes for antibiotic resistance.Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host. Plasmids can be found in all three major kingdoms, Archea, Bacteria and Eukaryote. Among eukaryotes, plasmids have been found in fungi and plants but not in animals. Most plasmids are mitochondrial. In filamentous fungi, plasmids are commonly encountered in isolates from natural populations. Individual populations may show a predominance of one type, but some plasmids have a global distribution, often crossing species boundaries. Circular plasmids are common only in Neurospora spp., but linear plasmids have been found in many fungi. Circular plasmids have one open reading frame (ORF) coding for a DNA polymerase or a reverse transcriptase. Linear plasmids generally have two ORFs, coding for presumptive DNA and RNA polymerases with amino acid motifs showing homology to viral polymerases.

19. Which DNA binding protein motif is present in Lac repressor : Which DNA binding protein motif is present in Lac repressor helix-turn-helix zinc finger homeodomain leucine zipper The helix-turn-helix motif consists of about 20 amino acids. The basic form is that of 2 helices separated by a turn. One helix, the stabilization helix, is there to stabilize the other helix, the recognition helix. The helix-turn-helix motif will be illustrated with the lac-repressor-DNA complex. The leucine zipper is an interesting structure made up of two a-helical segments of protein that have leucines facing each other along the length of the helices, allowing them to dimerize and form a symmetric interface that can bind to the DNA on both sides of the double helix (see figure). The leucine zipper motif will be illustrated with the GCN4 (protein)-AP1 (DNA) complex, a protein involved in activating transcription in yeast. The Zinc finger motif includes a metal, Zinc, in the DNA-binding motif. The Zinc helps to stabilize the three-dimensional structure of the Zinc-finger. The Zinc is coordinated either with the sulfur in cysteine, or one of the nitrogens in the histidine imidazole sidechain. The zinc-finger motif will be illustrated with the Transcription Factor IIIA-DNA complex from the clawed frog Xenopus laevis.

20. Which of the following gene is carried by "F" plasmid ? :. Which of the following gene is carried by "F" plasmid ? yrp yar tra trp lac Yrp - yeast replication plamid Yar – genes found in Human Y chromosome Trp – found in trp operon Lac – found in lac operon The Fertility factor is a bacterial DNA sequence that produces a sex pilus necessary for conjugation. It contains 20 tra (for "transfer") genes and a number of other genetic sequences responsible for incompatibility, replication, and other functions. The F factor is an episome and can exist as an independent plasmid or integrate into the bacterial cell's genome.

Slide 25 : F plasmid is responsible for oriV origin for vegetative replication -replication is bi-directional (like bacterial chromosome) -replicates in synchrony with bacterial chromosome oriT origin of transfer - rolling circle replication - single strand enters recipient which will synthesize the complementary strand Chromosomal Transfer by F-plasmid tra - transfer genes - genes on F-plasmid (tra genes) specify formation of sex pilus and conjugation bridge usually only F-plasmid is transferred,sometimes chromosomal genes are moved. http://highered.mcgraw-hill.com/olc/dl/120082/bio_f.swf visit this URL for the video.

21. In zinc finger Zn++ ion is co-ordinated to : . In zinc finger Zn++ ion is co-ordinated to 4 Cys 4 His 2 Cys and 2 His Both a and c The zinc finger motif contains one or more zinc ions which are crucial for the structural stability. It can be divided into three types: C2H2 zinc finger: It is characterized by the sequence CX2-4C....HX2-4H, where C = cysteine, H = histidine, X = any amino acid. In the 3D structure, two cysteine residues and two histidine residues interact with a zinc ion .Eg:S1 transcription factor C4 zinc finger: Its consensus sequence is CX2CX13CX2CX14-15CX5CX9CX2C. The first four cysteine residues bind to a zinc ion and the last four cysteine residues bind to another zinc ion. Eg Estrogen receptor C6 zinc finger. It has the consensus sequence CX2CX6CX5-6CX2CX6C. The yeast's Gal4 contains such a motif where six cysteine residues interact with two zinc ions. Eg: GAL4

Slide 27 : In Fig 2&3 Green strands – Cysteine residue Orange Balls – Zn ion Red Strands - Receptor Fig 2 Fig 3

22. Which one is not used in making genetically modified crops : . Which one is not used in making genetically modified crops vector based on plant virus various plasmid DNA artificial plasmid of animals natural plasmid of agrobacterium The agrobacterium transfers its own T-DNA, but if the T-DNA is removed and replaced with another gene, A. tumefaciens can be used to introduce that gene into the plant genome, thereby providing a vector for scientists to engineer beneficial genes into plants. Larger genes can be introduced to plants using bacterial artificial chromosomes (BACs). BACs are synthesized gene vectors based on a plasmid from E. coli. BACs can have inserts ranging from 50 to 350 kb, allowing for the transfer of large genes or many small genes at once. For specialized studies (production of foreign proteins in plants, expression of potentially lethal genes, etc.), virus-based vectors are used.

23. E.Coli cannot : E.Coli cannot degrade recombinant protein always fold recombinant protein correctly glycosylate recombinant protein correctly All of above Advantages of prokaryotic recombinant protein expression systems: Ease of culture Rapid cell growth Inducible expression using IPTG Simple purification Some of the disadvantages in using E.coli as the host are: Most proteins become insoluble and are very difficult to recover as functional proteins Post-translational modifications are not added by bacteria Protein may not be functional. http://www.exptec.com/Stategies/Problems%20and%20hosts.htm follow this URL for the expression problems due to hosts

24. Lac promoter can be induced by:- : Lac promoter can be induced by:- Iso propyl thio galactosidase iso methyl thio cyanate tryptophan 3-beta -indole acrylic acid Isopropyl-ß-D-thio-galactoside (IPTG) is frequently used as an inducer of the lac operon as it binds to repressor and inactivates it. Unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from "eating up" or degrading the inductant; therefore the IPTG concentration remains constant. IPTG can enter thye cells without lactose permease,and hence it can act as an inducer in the absence of functional lacy and lacz products

26. pBIN 19 binary vector consist of : pBIN 19 binary vector consist of lac z' gene kanamycin resistance gene cloning sites all of above

27. In lac operon the repressor is encoded by : . In lac operon the repressor is encoded by Lac A Lac Y Lac Z Lac I The operon is under the control of the adjacent lacI gene, encoding the lactose repressor. The repressor is a regulatory gene. In the absence of allolactose, the inducer of the lac operon, the repressor tetramer binds to the lac operator (lacO) and prevents RNA polymerase from transcribing the operon. However, when allolactose is present it binds to the repressor this prevents repressor from binding to lacO and permits RNA polymerase to bind to lacP and to initiate transcription. The lacI gene is transcribed constitutively from its own promoter. The lac repressor is a tetramer, where all four identical components are 360 amino acids in length. There are three main domains in the lac repressor molecule. The NH2 terminal domain (residues 1-62), the coreprotein domain (residues 62-340), and the COOH-terminal domain (residues 340-357) The NH2 terminal domain of the lac repressor is involved with the DNA binding. The NH2 terminal ends can actually be subdivided into two separate functioning domains: the DNA-binding region (residues 1-45) and the hinge region (residues 46-62) http://biology.kenyon.edu/BMB/Chime/Mat/MASTER.HTM follow the link for more details.

28. Drawback of using baculovirus in protein production : Drawback of using baculovirus in protein production larger nuclear inclusion bodies makes large amount of protein improper glycosylation polyhedrin gene product Advantages of using baculovirus expression system are Eukaryotic post-translational modification Proper protein folding and function High expression levels Easy scale up with high-density suspension culture Disadvantages of using baculovirus expression system are Low secretion level. Improper glycosylation. Proteins that occur as aggregates. Refrerence: http://books.google.com/books?id=QUd1FfElRN4C&pg=PA31&lpg=PA31&dq=polyhedrin+gene+product&source=web&ots=ge0SjQefy-&sig=2ebOC49QenqMOhdgiDqEXpZQ&hl=en&sa=X&oi=book_result&resnum=9&ct=result#PPA27,M1 Polyhedrin gene product is non essential when the virus Is propagated.In its absence BV is secreted by the infected cells. This dispensible nature of polyhedrin gene makes BV suitable for constructing vectors. The desired gene is placed under the control of polyhedrin promoter.

29. A yeast vector carrying A 2 ?m circlular origin of Replication : . A yeast vector carrying A 2 ?m circlular origin of Replication YAC YEp YIp YRp The YpI integrative vectors do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination.The YpI integrative vectors do not replicate autonomously, but integrate into the genome at low frequencies by homologous recombination. The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 mm plasmid that serves as an origin of replication (2 mm ori). The 2 mm ori is responsible for the high copy-number and high frequency of transformation of YEp vectors. The YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences, CEN, and autonomously replicating sequences, ARS. The YCp vectors are typically present at very low copy numbers, from 1 to 3 per cell. A yeast artificial chromosome (short YAC) is a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. The Yrp contain ARS as their origin of replication in yeast. they have very high copy numbers but are extremely unstable.

30. Factor Xa cuts at . Factor Xa cuts at After Arg of Gly-Arg Before Arg of Gly-Arg Before Gly of Gly0 Leu After Gly of Gly-Leu Factor Xa is a serine endopeptidase composed of two disulfide-linked subunits that converts prothrombin to thrombin in the blood coagulation cascade. Factor Xa cleaves after the arginine residue in its preferred cleavage site Ile-(Glu or Asp)-Gly-Arg and it will occasionally cleave at other basic residues. However, it will not cleave at a site followed by proline or arginine. Factor Xa is often used to remove fusion tags, such as the common histidine tag, from expressed proteins. By treating a purified, 6xHis-tagged protein expressed with a factor Xa cleavage site, it is possible to obtain the protein in its native form. The purified, expressed protein is incubated with factor Xa protease.

31. A.tumefaciens causes . A.tumefaciens causes rust wort crown gall disease hairy root disease Agrobacterium tumefaciens causes crown gall disease of a wide range of dicotyledonous (broad-leaved) .The disease gains its name from the large tumour-like swellings (galls) that typically occur at the crown of the plant, just above soil level.Crown gall tumors result from the over-production of the phytohormones auxin and cytokinin specified by A. tumefaciens T-DNA genes. wart:Synchytrium endobioticum, a primitive parasitic fungus forms wart.The primary symptom consists of galls on tubers, stolons and stems, and occasionally on leaves, but never on roots. The rusts belong to the Basidiomycota, the division of fungi to which the mushrooms belong. Although the rusts attack a large number of seed plants and even some ferns.The common name rusts is based on the "rusty" colored blotches that is present on the stems and leaves from the urediospore stage in this group of fungi.Eg:Late Blight of Potato disease was due to the fungal pathogen, Phytophthora infestans. Hairy root disease is caused by A.rhizogenes.Although the T-DNA of some Riplasmids of A. rhizogenes contains auxin biosynthetic genes, these loci are not always necessary for hairy root formation. Recent experiments suggest that hairy root tumors result from the increased sensitivity of transformed cells to endogenous auxin levels. 1 2 4 3

33. Which is not a method of DNA insertion in living cells : . Which is not a method of DNA insertion in living cells Transformation Transfection Microinjection none Transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of foreign genetic material (DNA). Terms used for genetic alterations resulting from introduction of DNA by viruses (transduction) or by cell-cell contact between bacteria (conjugation). Transformation of eukaryotic cells in tissue culture is usually called transfection. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transfection of animal cells typically involves opening transient pores in the cell plasma membrane, to allow the uptake of material. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Microinjection refers to the process of using a very fine needle to insert substances at a microscopic or macroscopic level into a single living cell. It is a simple process in which a needle 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the nuclear envelope and the desired contents are then injected into the desired sub-cellular compartment and the needle is removed.

34. Use of regulatory protein to protect DNA from endonuclease is done in . Use of regulatory protein to protect DNA from endonuclease is done in DNA footprinting DNA fingerprinting gel retardation chemical degradation DNA Footprinting was developed in 1977 and is an analytical procedure in molecular biology for identifying the specific sequence of DNA (the binding site) that binds to a particular protein. DNA Footprinting is most commonly performed on proteins that are thought to play some significant functional role such as gene regulation. DNA Fingerprinting, method of identification that compares fragments of deoxyribonucleic acid (DNA) It is sometimes called DNA typing. Gel retardation is a technique used to find out the DNA-protein interactions by electrophoresing the DNA fragment and the DNA-protein complex.The DNA protein complex moves slowly in the GEL which shows the presence of the protein. Maxam Gilbert method requires radioactive labelling at one end of the DNA. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA fragment, from which the sequence may be inferred.


35. Advantage of insulin production by recombinant DNA technique : . Advantage of insulin production by recombinant DNA technique can be modified by addition of sugar molecules after translation insulin is a big protein active insulin is synthesized by bacteria none Human insulin is the only animal protein to have been made in bacteria in such a way that its structure is absolutely identical to that of the natural molecule. This reduces the possibility of complications resulting from antibody production. In chemical and pharmacological studies, commercially available Recombinant DNA human insulin has proven indistinguishable from pancreatic human insulin. The only disadvantage was contamination due to host cell and it requires thorough purification. Nowadays, the recombinant insulin is produced using Yeast cells as they secrete an almost complete human insulin molecule with perfect three dimensional structure. This minimises the need for complex and costly purification procedures.



36. trp promoter is repressed by . trp promoter is repressed by leucine lactose galactose Tryptophan The trp operon is a repressible system. The primary difference between repressible and inducible systems is the result that occurs when the effector molecule binds to the repressor. With inducible systems, the binding of the effector molecule to the repressor greatly reduces the affinity of the repressor for the operator, the repressor is released and transcription proceeds. The lac operon is an example of an inducible system. With repressible systems, the binding of the effector molecule to the repressor greatly increases the affinity of repressor for the operator and the repressor binds and stops transcription. Thus, for the trp operon , the addition of tryptophan (the effector molecule) to the E. coli environment shuts off the system because the repressors binds at the operator.



37. T-DNA is not T-DNA is not between 15-30 Kb in size passed to daughter cells responsible for cancerous properties responsible for opine synthesis During tumour induction, Agrobacterium attaches onto plant cells and then transfers part of its DNA to some of these cells. The transferred DNA (T-DNA) which is found on a large Ti (tumour inducing) plasmid is modified within the bacterium and is transferred to the plant where it becomes integrated into the plant genome. Proteins which are encoded by the virulence (vir) region of the Ti plasmid regulate the T-DNA modification and transfer. Expression of genes located on the T-DNA leads to the formation of proteins involved with the production of auxins and cytokynines. These plant hormones cause the tumourous phenotype that is characterised by the ability of the plant cells to proliferate limitlessly and autonomously, even in the absence of added phytohormones. Crown gall tumours are characterised by the production of opines (derivatives of amino-acids). The biosynthesis of opines is catalysed by opine synthase enzymes, which are encoded by the T-DNA. Opines are formed in the tumours can be metabolised by the tumourigenic agrobacteria, but not by most other soil organisms. Thus, Agrobacterium creates for itself a favourable niche by genetic modification plant cells.

38. which one will affect efficiency of expression study of foreign gene in Ecoli . which one will affect efficiency of expression study of foreign gene in Ecoli presence of introns Sequence acting as termination signals in E.Coli codon bias All of above Introns: The foreign gene might contain introns.Since the E.coli does not have introns the host would not have proper machinery to remove introns from transcripts. The foreign gene might also contain sequences that act as termination signals in E.coli.These sequences might be completely innocuous in normal cell but in E.coli results in premature termination and loss of gene expression. Codon bias: tRNA availabilty is addressed,the highly expressed genes will have codons corresponding to abundant tRNAs and the genes expressed at low levels use codon for less abundant tRNA. Equalization of codon-anticodon hydrogen bonding. In cases where there is a choice of synonymous codon,selection would be to avoid very strong and weak interaction with anticodon,which decreases the translation efficiency. A variety of factors affect the expression of foreign proteins in Escherichia coli. These include: promoter strength, efficiency of ribosome binding, stability of the foreign protein in E. coli, location of the foreign protein in E. coli, the codons used to encode the foreign protein, the metabolic state of the cell, and the location, stability and copy number of the foreign gene.

39. Promoter used for control of cloned genes in saccharomyces cerevisiae Promoter used for control of cloned genes in saccharomyces cerevisiae gal pho5 cup1 All of above Cloned genes in S. cerevisiae are placed under the control of GAL promoter which is normally upstream of the gene coding galactose epimerase (an enzyme for galactose metabolism). The GAL promoter is induced by Galactose. PHO5 promoters are regulated by the phosphate level in growth medium. CUP1 is induced by copper.

40. Repression of lac operon is not caused due to Repression of lac operon is not caused due to absence of lactose mutation in operator functional I gene on it functional I gene on another DNA in cell cis-acting locus - a genetic region affecting the activity of genes on that same DNA molecule Such a locus usually does not code for a protein but instead acts as a binding site for trans-acting proteins. Jacob and Monod proposed the "operator element" in the lac operon. - If mutated this operator element should be dominant in cis in that it only affects the genes on the same chromosome (directly adjacent to it). - It will not be dominant in trans. OC is dominant in the cis position - no diffusible product. cis dominance - the ability of locus to influence the expression of one or more adjacent loci on the same chromosome, as occurs in lac operator mutants of E.coli

41. which one is not encoded by structural gene of lac operon : . which one is not encoded by structural gene of lac operon ß-galactosidase galactosidase acetylase galactosidase permease thio-galactosidase transacetylase lacZ encodes ß-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. lacY encodes ß-galactoside permease, a membrane-bound transport protein that pumps lactose into the cell. lacA encodes ß-galactoside transacetylase, an enzyme that transfers an acetyl group from acetyl-CoA to ß-galactosides

42. Which of the phage never cause cell lysis? . Which of the phage never cause cell lysis? M13 Lambda F-X174 none Bacteriophage lambda has two different life cycles. It can infect its host, E. coli, replicate and synthesize new phage, then lysing and killing the host as the phage burst from the cell. Or, the phage can infect the cell and enter a dormant phase within the cell in which the phage is present but its genes are generally not being expressed. Then under certain circumstances, the phage will leave dormancy and resume lytic growth. Coliphage F-X174 encodes a single lysis protein, E, a 91 amino acid membrane protein,which is a specific inhibitor of Mra Y gene involved in peptidoglycan synthesis. Filamentous hages like M13 and Ff are not lytic.Some of these phages become prophages by integrating into the genome,others remain as episomal or plasmid like prophages and establish a pseudolysogenic relationship with host,when they do not integrate but continuously produce progeny.

43. Examples of regulon are . Examples of regulon are SOS response to DNA damage heat shock gene system sugar metabolism system All of above In eukaryotes, a genetic unit consisting of a noncontiguous group of genes under the control of a single regulator gene. In bacteria, regulons are global regulatory systems involved in the interplay of pleiotropic regulatory domains and consist of several OPERONS. When bacteria are exposed to stress they can produce many defence proteins which genes are normally in a repressed state and that allow repair of damaged DNA and reactivation of DNA synthesis, and that these processes are connected with mutation. Since emotionally he was bound with the sea he called this phenomenon the SOS response, after Save Our Souls. Heat shock regulons contain a group of heat shock proteins (stress proteins). They are induced when a cell undergoes various types of environmental stresses like heat, cold and oxygen deprivation. For example, HSPs help new or distorted proteins fold into shape, which is essential for their function.

44. In HSP which s subunit mediates RNA polymerase binding to promoter : . In HSP which s subunit mediates RNA polymerase binding to promoter s70 s32 s54 s90 Different ss have different affinities for variant promoters. * s70 - general purpose. * s54 - nitrogen metabolism. * s32 - heat shock proteins. * sS - stationary phase. * sF - flagella.

45. What is the function of Alkaline phosphatase and polynucleotide kinase : . What is the function of Alkaline phosphatase and polynucleotide kinase Removes phosphate group from 5' end and add at 5' end respectively Removes phosphate from 3' end and adds at 3' end respectively Adds phosphate at 5' end and removes from 5' end respectively Adds phosphate at 3' end and removes from 3' end respectively Alkaline phosphatase removes 5' phosphate groups from DNA and RNA. It will also remove phosphates from nucleotides and proteins. These enzymes are most active at alkaline pH - hence the name. Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate from ATP to the 5' end of either DNA or RNA.

46. Similarity between chain termination and chemical degradation method of sequencing : . Similarity between chain termination and chemical degradation method of sequencing use double stranded DNA Need Primer Degrades the DNA none See Next slide for the explanation. use double stranded DNA – maxim gilbert method (chemical degradation method) Need Primer – Sanger method (chain termination method) Degrades the DNA - (chemical degradation method)



47. which will negatively effect lac operon expression : . which will negatively effect lac operon expression lactose less glucose concentration reased binding of cAMP and CRP decrease in concentration of c-AMP The activity of RNA polymerase also depends on the presence of another DNA-binding protein called catabolite activator protein or CAP.CAP can bind to DNA only when cAMP is bound to CAP. so when cAMP levels in the cell are low, CAP fails to bind DNA and thus RNA polymerase cannot begin its work, even in the absence of the repressor.


48. s subunit is a type of . s subunit is a type of specificity factor repressor activator none Bacterial RNA polymerases are best characterised in Thermus aquaticus. The five subunits are: * aI aII - required for DNA binding and assembly. * ß ß' - needed for DNA binding (contains basic amino acid residues) and catalysis. * ? - stabilises ß' binding. * s - forms holoenzyme. s stimulates tight binding between RNA polymerase and a promoter.Hence it is the specificity factor. A repressor is a DNA-binding protein that regulates the expression of one or more genes by decreasing the rate of transcription. An activator is a DNA-binding protein that regulates one or more genes by increasing the rate of transcription.

49. Vector used in first cloning experiment in mammalian cell was based on Vector used in first cloning experiment in mammalian cell was based on SV 40 BPV adenovirus retrovirus Animal virus vectors also deliver the foreign genes into the cultured cells which get integrated into the host genome. The expression of foreign genes can also be amplified using the promoters of the virus gene. The cloned genes can be used in gene therapy, for the synthesis of important proteins etc. A vector based on Simian Virus 40 (SV 40) was used in the first cloning experiment involving mammalian cells in 1979. SV40 is capable of infecting several mammalian species following a lytic cycle in some hosts and lysogenic in some. Adenovirus are bigger viruses and are capable of cloning 8KB fragments. BPV Bovine papilloma virus has an unusual infection in mouse cells taking the form of multicopy plasmid with about 100 copies per cell. BPV molecules are passed on to daughter cells.They do not cause death in mouse.

50. Full form of HART is : Full form of HART is Hybridization and retranslation Hybrid arrest translation Hybridization and retsnscription High affinity restriction technique

51. HRT and HART depend on . HRT and HART depend on bacterial cell translation eukaryptic cell translation both prokaryot and eukaryotic cell translation cell free translation Both HRT and HART rely on hybridising cloned DNA fragments to mRNA of the cell or tissue from which the clones have been derived.In both cases the identity Protein, hence the gene can be determined by examining the gels.



52. Shotgun cloning is appropriate for Shotgun cloning is appropriate for prokaryotes eukaryotes both none Prokaryotes contain complete and uninterrupted ORFs - therefore prokaryote genes can be cloned directly from genomic DNA. Most eukaryotes have ORFs which are divided into coding (exon) and non-coding (intron) sequences – therefore these genes cannot be cloned directly from genomic DNA - these require cDNA cloning

Parasitic Protozoan Imp topic for Various Life Science Examination 2010

Parasitic Protozoans
[A] Entamoeba dysenteriae (=histolytica)

1. Lambl discovered Entamoeba in 1859. Losch discovered its pathogenic nature in 1875.
2. It is Monogenetic Endoparasite of the large intestine (colon) of man and other mammals.

Pathogenesis:
The adult Trophozite or Magna form secretes histolytic enzymes which dissolve and damage the intestinal wall producing ulcers in it. Blood and mucus comes out of the ulcers and passes out along with the faeces of the patient. This disease is known as amoebic dysentery or Amoebiasis.
Life Cycle:
1. Inside the ulcers, the adult tropozoite divides repeatedly by reproducing by Binary Fission to produce 2 types of individuals: Magna forms (large sized) and Minuta forms or Pre-cystic forms (small-sized).
2. Magna forms remain inside the ulcers and keep on reproducing by binary fission but minuta forms come out of the ulcer and undergo following changes:
(a) Withdraw pseudopodium and become rounded.
(b) Store reserve food in the form of chromotoid bodies.
(c) A cyst is secreted i.e. Entamoeba becomes encysted (Encystment).
(d) Nucleus divides twice by mitosis to form 4 nuclei.
3. These Quadrinucleate cysts pass out along-with the faeces. These are infective stages for new a host.
4. Cysts are spread by houseflies, ants cockroaches etc. which carry them on their appendages or in their intestine and transfer them to the foodstuffs.
5. Man gets the infection by swallowing food and water contaminated by quadrinucleate cysts (Quadrinucelate cysts remain viable for 8-10 days. They die due to desiccation/or at a temperature of 50oC).
6. In man's small intestine the cystwall is digested by Trypsin and the Tetranucleate Metacystic amoeba hatches out (Excystment).
7. Metacystic Amoeba divides by binary fission to produce Eight uninucleate daughters/amoebulae which pass into the large intestine and grow into adult trophozoites.
Therapy:
1. Common drugs are injections of Emetine; Tablets of antibiotics e.g. Fumagilin, Terramycin, Erythromycin and Aureomycin are quite effective. For the complete eradication of infectin when dysentery has been controlled, certain Arsenic compounds (Carbarsone, Milibis) and iodine compounds (Chiniofon, Diodiquin, Vioform) are given in small doses.
2. In Chronic infection of Entamoeba, the trophozoites escape into the general blood circulation of the body causing secondary infection in Liver, Lung, Brain etc. producing abscesses in these organs. For these Extra-Intestinal infections Chloroquine is given.
3. Most significant advancement in the treatment of Amoebiasis has been the use of Metronidazole which has been found to be quite effective in case of both intestinal and extra intestinal infection. Some of the popular Anti-amoebic drugs are Entobex, Diogyl, Amibactin, Dependal, Enteroquinol, Flagyl, Metrogyl, Tridazole etc.


[B] Entamoeba gingivalis

Found in the roots of teeth, gums, pus-pockets of Tonsils of Man & other mammals. Cytoplasm clearly divided into Ectoplasm & Endoplasm; Pseudopodia 2 to 3; Feeds upon Bacteria found in the debris and White Blood Corpuscles; Transmission is direct through saliva & kissing; No cyst formation occurs. It aggravates the already present Pyorrhoea.

[C] Entamoeba coli

Found in the lumen of the human colon; Cytoplasm not clearly divided into Ectoplasm & Endoplasm; Pseudopodium-one; Feeds upon Bacteria and debris; Man is neither benefited nor harmed by its presence (COMMEMSAL); Transmission through Eight Nucleate Cysts; No further nuclear division occurs in the Cyst.

TRYPANOSOMA
(A Flagellate Protozoan)

A saprozoic blood parasite of man & domestic animals causing "Trypanosomiasis". It slowly glides or swims in the plasma by vibratile movements of its Flagellum and undulating membrane. Feeding occurs by diffusion through pellicle and by pinocytosis. Trypanosomes reproduce only asexually by Longitudinal Binary Fisson in both the vertebrate and invertebrate hosts. The reserve food of Trypanosoma consists of Volutin Granules (Glycogen/Protein/Nucleic Acid). Trypanosoma is a Polymorphic Protozoan having Trypanosome (Trypanomastigote); Crithidial (Epimastigote); Leptomonad (Promastigote); and Leishmania (Amastigote) forms in its life-cycle. Crithidial forms are found in the salivary glands of Tse-tse fly.

1. T. gambiense- West & Central Africa2. T. rhodesiense- East Aftrica The primary host is man (Homo) Intermediate or Vector hostTse-Tse fly (Glossina palpalis)Tse-Tse fly (Glossina morsitans)

Sleeping Sickness:
When Tse-Tse fly bites a man for its blood-meal it pours a drop of saliva containing anticoagulant. Alongwith the saliva Tryanosomes are inoculated into the blood of man. For about three months, trypanosomes live in the Blood-plasma and Lymph glands causing fever (Gambian – Fever) & anaemia. The sleeping sickness is caused when parasites invade cerebro-spinal fluid and brain-cells resulting in extensive damage to the C.N.S. The patient wants to sleep constantly, finally passing into a state of COMA. If untreated, it ends in death. Tryparsamide and Antryside are used in the treatment of sleeping sickness.
3. Trypanosoma cruzi = causes CHAGAS' DISEASE:
In Central and South America; being tranmitted by blood-sucking Triatomid Bugs (Kissing Bugs – Triatoma & Rhodnius) which serve as Intermediate hosts. Man gets the infection when its skin (exposed wounds, conjunctiva) comes in contact with faeces of the bug containing Trypanosomes. It is an intracellular parasite of the tissues of heart, voluntary muscles, C.N.S. and glands. Chagas' disease is characterized by fever, anaemia and disturbances of C.N.S. It is more common in infants and children.
4. Trypanosoma evansi causes "SURRA" disease of cattle in Africa, Asia & S. America. Similarly, Trypanosoma brucei causes "NAGANA" disease of cattle in Africa. These are also digenetic parasites being transmitted by blood sucking Dipteran flies. T. lewisi is found in Rats in North America. It is non-pathogenic and is transmitted by Rat-flea.
LEISHMANIA
(A Flagellate Protozoan)

1. Leishmania donovoni:
Causes Kala-azar (Dumdum Fever) or Visceral Leishmaniasis in Man in Africa, India, China, & America. Parasites attack endothelial cells of blood vessels, Lymphatics, Spleen, Liver, Bone-marrow and eventually Leucocytes of blood. Parasites are found both outside and inside the tissue cells but they are less numerous in circulating blood. The symptoms include fever, anaemia, enlargement of spleen and liver. The Intermediate or Vector hosts are blood-sucking Sandiflies (Phlebotomus sp.) in whose proboscis they are stored.
2. Leishmenia tropica:
It causes a human skin-disease known as "Oriental Sore" or Delhi Boil or Cutaneous Leishmaniasis in India, Africa, Russia, Torpical America. Parasites are found in the endothelium of skin capillaries and dermal tissues of sores. Sores cause ulcerating wounds which take several months to heal. The disease is transmitted by the blood-sucking Sandiflies or by direct contact.

3. Leishmania brasiliensis:
Cause "Espundia or Bubos" or Naso-oral Leishmaniasis in Americans. The insect vector is sandfly, in this case too.

Some Other Pathogenic Protozoans

Name of the Parasite Transmission Disease
1. Trichomonas vaginalis(Flagellate) Monogentic, Direct through sexual intercourse. Inflammation of mucous-membrane of vagina in women (vaginitis) and Urethra in men, leading to Leucorrhoea.
2. Trichomonas hominis Monogenetic, Transmission through infective cysts in faeces. Large intestine of man and other mammals, associated with Diarrhoea, and Dysentery.
3. Trichomonas tenax Monogenetic, Transmission direct through kissing T. tenax and E. gingivalis are found in pus pockets formed between teeth and the gum in the disease Pyorrhoea. They probably aggravate the disease.
4. Giardia – (Lamblia) intesinalis (Flagellate) Monogenetic, Transmission through infective cysts in faeces. Small intestine of man, associated with Diarrhoea. (Giardiasis)
5. Balntidium coli (a ciliate) Monogenetic, Transmission through cysts. Large intestine of Man, Monkey, pigs etc. causes ulcerations in colon resulting in Balantidial Dysentery.



MALARIAL PARASITE

IMPORTANT SCIENTISTS :-
1. LANCISI in 17th century first suspected a relationship between Malaria and Mosquito.
2. CHARLESS LAVERAN (1880) discovered that Malaria is caused by a protozoan parasite, Plasmodium. He was the first to have observed various stages of plasmodium in Man’s blood. CELLI (1885) supported Lavern’s observations. GOLGI (1885) made a detailed study of blood cycle.
3. SIR RONALD ROSS(1898) was the first to observe oocysts on the stomach of Anopheles (Nobel Award 1902).
4. GRAASSI (1898), for the first time described the Life-Cycle of Plasmodium in Mosquito
5. SHORTT & GARNHAM (1948) studied Pre-Erthrocytic and Exo-Erthrocytic cycle.

1. P. falciparum : = 9-12 days
2. P. vivax : = 10-14 days
3. P. ovale : = 10-14 days
4. P. Malariae : = 28-30 days

PRODROMAL SYMPTOMS: Symptoms appearing before the actual attacks of malarial fever are Nausea, Loss of appetite, Constipation, Insomnia, Mouth becomes dry, Headache and Bodyache.

PAROXYM (Fits of Fever): The actual attack of Malarial fever whose duration is about 6 hours includes 3 stages:
I. Rigor Stage : Chilling & Shivering
II. Febrile Stage : High fever 1040 to 1050 F
III. Defervescent Stage : Profuse sweating brings down the temperature

TIME REQUIRED FOR THE COMPLETION OF ERYTHROCYTIC CYCLE
1. In P. falciparum, P. vivax and P, ovale the erythrocytic cycle is completed in 48 hours, hence the fever comes every third day.
2. In P. malariae, the erythrocytic cycle is completed in 72 hours hence the fever comes every 4th day.

TYPES OF MALARIA
A. Tertian Malaria: Paraoxym every third day. Tertian malaria is of 2 types:-
(a).Benign Tertian : Mild Malaria caused by P. vivax & P. ovale. Death rates is low because merozoites destroy old and mature RBC’s.
(b). Malignant Tertian: Fatal malaria caused by P. falciparum, death rate is high because infected RBC’s usually clump together and block capillary blood circulation in vital organs like brain, lungs, heart etc. Because of large scale destruction of RBC’s (Pernicious Malaria) haemoglobin is excreted in urine (Black water fever).

B. Quartan Malaria: Paroxysm every 4th day; caused by P.malariae. Large scale destruction of young RBC’s might lead to secondary complications.

C. Quotidian Malaria: Paroxysm irregular or daily, caused by mixed species infection.

1. P.OVALE is the rarest of the four speceies of Malaria parasities. P.ovale is mostly restricted to west Africa P. Vivax is the commonest species being prevalent in both tropical and temperates Parts of the world. P. malariae is also found in both tropical and tempreture climates but it is less common than P.vivax. P. falciparum is very common in tropical countries (Tropical Malaria)

2. P. falciparum causes the most dangerous kind of malaria called Malignant tertian of Aestivo-autumnal malaria Since erythrocytic cycles are completed in 24 to 48 hours this is also knows as Sub-tertian Malaria.
In falciparum infection, RBC’s clump together and prevent proper flow of blood into `the brain & other vital orans. Because of this and the toxic effect of the parasite, the patient suffers from total loss of consciousness (Coma) and sometime sudden death occurs. this Cerebral Malaria “is responsible for thousands of malarial deaths ;

Effects of Malaria on Human being:

1. Anaemia: (Shortage of Haemoglobin) due to large Scale destruction if RBC’s
2. phagocytic cells of Reticulo- endothellal system found in the Spleen and Liver phagocytize and remove the infected RBC’s and merozoites from the blood circulating through them . These organs produce more and more phaocytic cells resulting in their own enlargement. Enlarged spleen probably releases Lysolecithin which further destroys RBC’s Toxic substances & pigments are deposited in the liver, spleen and under the skin.
3. Because of the large scale break down of RBC’s large quantities of Bile pigments (Bilirubin & biliverdin) are produced which gradually accumulate in blood giving the skin, mucous membrances,a yellow colour causing Jaundice.

Therapy/Treatment of Malaria:

Quinine is the oldest drug obtained from the bark of Cinchona tree of family Rubiaceae. it destroy all the stages of the parasite in patients blood .Atebrin (mepacrine) camoquin chloroquine are similar to quinine. Paludrine pentaquine Daraprim and camoprima are other anti malarial drugs which destory both exo-erythrocytic stages of the parasite.
Halofantrine has been found to be quite effective against falciparum infection Artemesin is a new anti –malarial drug, obtained from the plant Artemesia annua, Family compositae (developed by CIMAP,Lucknow). Artemesin is effective against cerebral Malaria,

Control of Malaria

(A) Destruction of Anopheles;

1. Adults can be destroyed by using insecticides like DDT , B.H.C, Pyrethrum etc.Fumigants such as Sulphur,Tarcamphor or derivatives of Naphtha are burnt to produce poisonous fumes that kill the mosquitoes.
2. Destruction of breeding places by maintaining proper drainage and not allowing the stagnation of the water.
3. Destruction of larvae & pupae by spraying oil on the water surface, thus preventing them from carrying out their normal respiration. The Biological Control lies in producing natural enemies of mosquito larvae, pupae into the ponds such as fishes (Gambusia; Lebistes Trouts Minnows; Stickle- Backs) Ducks insectivorous plants like Utricularia Droseraa etc.
4. Introduction of larvicidal Bacillus sphaericus in water bodies (Rajamohan et al, 1992).
5. Introduction of Blue Green Algae like Aulosira & Anabaena in water bodies.
(B) Prevention of infection (Prophylaxis)
1. mosquito can be prevented from biting by using mosquito nets and using mosquito repellents.
2. prophyactic drugs such as quinine and chloroquine if taken daily in small quantities , suppress malaria.

Relapse of malaria:
Exo- erythrocyties cycles may revive after a period of dormancy in case of P.vivax P. malariae and P ovule infection after the disease has been completely cured merozoites produced by these cycles attack RBC’s and may cause a Relapse of malaria.

· in case of P. falciparum infection greenish Maurer’s dots; in the infection of P. malariae Zeimann’s dots and in the infection of P. ovale james dots are formed in the cytoplasm of RBC’s .
· The duration of Mosquito–cycle is 10-17 days in P.vivax ; 16 days in P. ovale 22 days in P. falciparum and about 25-30 days in P. malariae.
· N.M.E.P. (National Malaria Eradication Programme) was started in India in 1953.
· P.berghei is found in rats & P. gallinacum is found in chickens.
· August 20 is celebrated as malaria Day. August 29-Mosquito Day

Plasmodium

1. Malaria is caused by a protozoan parasite, plasmodium and it is transmitted by Female Anopheles mosquito.
2. Life –cycle of Plasmodium is DIGENETIC i.e. two types of hosts are required:-
i).primary or Definitive or principal host is man.
ii).Secondary or intermediate or carrier or vector host is female anopheles mosquito.
Common species of Anopheles spreading malaria in our country are--- A. culicifacies, A. fluviatilis A. stephensi A. maculatus etc. plasmodium can not be transmitted by other species of mosquito because of its host- specificity.
Monkey serves as a Reservoir host for plasmodium. Reservoir host for leprosy
3. Plasmodium is pathogenic to man but ‘non pathogenic ‘to mosquito.




1 Sickle –shaped Sporozoites are infective stages of the Parasite for man. Thousands of sporozoites are inoculated into the man’s blood during mosquito bite.
2. Sporozoites disappear from blood-stream within half an hour or so. They dissolve the capillaries and enter into the liver cells with the help of histolytic enzymes secreted by a pair of secretary organelles.





1. Soon after its entry in the liver-cell, the sporozoite changes into a CRYPTOZOITE which grows to become a schizont. The schizont undergoes schizogony (a type of asexual reproduction by multiple fission) producing large number of CRYPTOMEROZOITES (100 to 1000).

2. Schizont and the liver cell burst releasing cryptomerozoites in blood sinusoids of Liver from where they escape into the blood circulation of the body to start Erythrocytic Cycle.

3. This is the first exo-erythrocytic cycle which is also known as Pre-Erythrocytic cycle. The time required for the completion of Pre-Erythrocytic Cycle is known as Pre-Patent Period (about 8-10 days)
4. Exo-erythrocytic cycle is absent in Plasmodium falciparum.

.


1 Some of the cryptomerozoiteis of the pre –erythrocytic cycle reinvade fresh liver –cells and change into Metacyptozoites (or phanerozoites) they are of two types ;
Micrometacryptozoites and macrometacryptozoites producing Micrometacryptomerozoites (about 1000) and Macrometacryptomerozoites (about 64) respectively by Schizogony.

2. Macrometacryptomerozoites reinvade fresh liver cells to continue the exo-erythrocytic cycles but Micrometacryptomerozoites attack RBC’s (just like the cryptomerozoites of the pre- erythrocytic cycle) to participate in the Erythrocytic cycle.




1. This cycle is started by merozoites (first of all by cryptomerozoites and
then by micrometacryptomerozoites)

2. After entering into the RBC the merozoite change into a young Trophozoite it develops a non – contractile vacuole which pushes the nucleus and cytoplasm toward the periphery giving it a ‘SIGENT-RING’ appearance.

3. With the help of its lytic enzymes the trophozoite feeds upon the cytoplasm of RBCs and grows to become AMOEBOID. it breaks haemoglobin to Globin (digestible) and Haematin (undigestible). Heamatin granules get deposited in the cytoplasm of the trophozoites in the form of brownish black toxic pigment called Haemozoin. Meanwhile yellowish orange granules of unknown nature called Schuffner’s Dots appear in the cytoplasm of the host RBC.

4. The Adult Inracellular Trophozoite. of plasmodium is now know as schizont which undergoes schizogony to produce about 24 merozoites or schizozoites.schizont and the host RBC burst . Ruptured RBCs are referred to as ghost cells. merozoites, haemozoin garnules and some other toxic substances are released in the blood plasma. it marks the end of the Erythrocytic schizogony and at this stage; the patient gets the characteristic attack of malarial fever.

Attack of Malarial Fever

5. The interval between the inoculation of sporozoits into the man’s blood and first attack of malarial fever is called INCUBATION PERIOD.
6. Erythrocytic cycle is completed in fixed time of either 48 hrs or 72 hrs., depending upon the species of plamodium. that is why the malaria patient gets the attack of fever each time the erythrocytic cycle is completed.
7. Most of the schizozoties re-enter into the RBC to continue the erythrocyte for increasing the number of schizozoites in the blood .However, some of the schizozoites attack RBC to become Gametocytes or GAMONTS which are of 2 types: Microgamonts and Megagamonts.
8. No further change is possible inside the body of man because he is warm-blooded. Gamonts need low temperature for developmeny hence they come in peripheral circulations of man between 10 pm to 2am When the female mesquites a malarial patient in the night she sucks the gamonts. Thus, Gamonts are ingective for the intermediate host (Mosquito).
9. If gamonts are not transferred into the body of the mosquito within few hours after their formation then they stages and disintegrate in man’s blood.



A. SEXUAL CYCLE OR GAMOGONY
1. Gamonts are resistant, hence they can not be digested inside the stomach of Mosquito. Here they undergo gametogenesis (Sexual-Cycle) to produce gametes.
2. Microgamont produces 6-8 microgametes or sperms by Exflagellation.On the other hand, Megagamont produces only one megagamete produces only one megagamete or ovum by Oogenesis. Ovum develops a cone of reception for receiving the microgamets.
3. Fertilization of a macrogamete by a microgamete results in the formation of a nonmotile spherical ZYGOTE in the lumen of the stomach of mesquite. There is a nuclear as well as cytoplasmic-fusion, hence it is called SYNGAMY. Since the fusing gametes are dissimilar, this syngamy is Anisogamous.
4. Now the Zygote becomes elongated, motile and worm-like to be know as OOKINETE.
5. Ookinete penetrates the stomach wall until it comes on the surface of the stomach. Zygote becomes encysted and now is called Oocyst.
B.SPOROGONY OR SPORE FORMATION:
1. The Oocyst grows to become SPORONT. It's nucleus divides repeatedly to form SPOROBLASTS or ZOITOBLASTS.
2. Sporoblasts then multiply and give rise to sporozoites which get arranged in a spokelike fashion. About ten thousand sporozoites are produced from a single Oocyst. A mosquito may have 50 to 500 Oocysts on its stomach.
3. Sporozoites swim in the haemolymph and then enter into the salivary glands where they are stored to be inoculated into the primary host i.e. man.
Ø Attempts have been made to develop vaccines against vulnerable malarial parasite stages using dead or attenuated parasites, but without success.
Ø One possible reason is that the antigen of the parasite may change over time, so that antibodies against the original antigens fail to recognize the new antigens.
Ø In the 1960 it was demonstrated that resistance to P. falciparum among west Africans was associated with the presence of Hb-s in their RBC
Ø Hb-s differ from normal Hb-A by a single amino acid, Valine in each half of Hb molecule, consequently these RBC responsible for sickle cell disease have a low binding capacity for oxygen. Because the malarial parasite has a very active aerobic metabolism, it can not grow and reproduce within thee RBC.