SDS PAGE - What each chemical ingredient does?

The importance of Poly Acrylamide Gel electrophoresis in protein research is un questioned. The composition of the gel is complex and the procedure is more an art than science. Precision is the key word in its making. This article provides information regarding the importance of various ingredients of the PAG

Poly acrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1954. Two independent groups Davis and Raymond employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that made it a versatile medium. Poly acrylamide gel separates protein molecules according to both size and charge. It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes, can withstand high voltage gradients, feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording. DISC electrophoresis utilizes gels of different pore sizes. The name DISC was derived from the discontinuities in the electrophoretic matrix and coincidentally from the discoid shape of the separated zones of ions (Anbalagan, 1999). There are two layers of gel namely staking gel or spacer gel and resolving gel or separating gel.
Staking gel or spacer gel: It is a large pore poly acrylamide gel (4%). This gel is prepared with Tris buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer. These conditions provide an environment for Kohlrausch reactions, as a result, proteins are concentrated to several fold and a thin starting zone of the order of 19 microns is achieved in a few minutes. This gel is cast over the resolving gel. The height of the staking gel region was always maintained more than double the height and the volume of the sample to be applied.
Resolving gel or Separating Gel: This is a small pore polyacryl amide gel (3 - 30%). The Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size. In the present experiment, 8%, 10% and 12% Resolving gel were used for separating different range of proteins. 8% gel for 24 – 205 kD proteins, 10% gel for 14-205 kD proteins and 12% gel for 14-66 kD proteins
The chemical ingredients of the gel are the following
Tris (tris (hydroxy methyl) aminomethane) (C4H11NO3; mw: 121.14). It has been used as a buffer because it is an innocuous substance to most proteins. Its pKa is 8.3 at 20°C and reasonably a very satisfactory buffer in the pH range 7.0 – 9.0.
Glycine (Amino Acetic Acid) (C2H5NO2; mw: 75.07). Glycine has been used as the source of trailing ion or slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative charge in the pH range. The minimum pH of this range is somewhere around 8.0.
Acrylamide (C3H5NO; mw: 71.08). It is a white crystalline powder. While dissolving in water, autopolymerisation of acrylamide takes place. It is a slow spontaneous process by which acrylamide molecules join together by head on tail fashion. But in presence of free radicals generating system, acrylamide monomers are activated into a free-radical state. These activated monomers polymerise quickly and form long chain polymers. This kind of reaction is known as Vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires hooking various chains together. Acrylamide is a neuro toxin. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.
Bisacrylamide (N,N’-Methylenebisacrylamide) (C7H10N2O2; mw: 154.17). Bisacrylamide is the most frequently used cross linking agent for poly acryl- amide gels. Chemically it is thought of having two-acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide was preserved at 4°C.
Sodium Dodecyl Sulphate (SDS) (C12H25NaO4S; mw: 288.38). SDS is the most common dissociating agent used to denature native proteins to individual polypeptides. When a protein mixture is heated to 100°C in presence of SDS, the detergent wraps around the polypeptide backbone. It binds to polypeptides in a constant weight ratio of 1.4 g/g of polypeptide. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment becomes a rod like structure possessing a uniform charge density, that is same net negative charge per unit length. Mobilities of these proteins will be a linear function of the logarithms of their molecular weights.
Ammonium per sulphate (APS) (N2H8S2O8; mw: 228.2). APS is an initiator for gel formation. APS was stored at 4°C.
TEMED (N, N, N’, N’-tetramethylethylenediamine) (C6H16N2; mw: 116.21). Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium per sulphate and the quarternary amine, N, N, N’, N’-tetramethylethylenediamine (TEMED). The rate of polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED. Increasing the amount of APS and TEMED results in a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in gel elasticity. Decreasing the amount of initiators shows the reverse effect. It is recommended that lowest catalysts concentrations that will allow polymerisation in the optimal period of time should be used. APS and TEMED are used, approximately in equimoloar concentrations in the range of 1 to 10 mM. TEMED was stored at 4°C.
The following chemicals are used for processing of the gel and the protein samples visualized in it.
Bromo Phenol Blue (BPB) (3’, 3’’, 5’, 5’’-Tetrabromophenolsulph- onephthalein) (C19H10Br4O5S; mw: 669.99). BPB is the universal marker dye. Proteins and nucleic acids are mostly colourless. When they are subjected to electrophoresis, it is important to stop the run before they run off the gel. BPB is the most commonly employed tracking dye, because it is viable in alkali and neutral pH, it is a small molecule, it is ionisable and it is negatively charged above pH 4.6 and hence moves towards anode. Being a small molecule it moves ahead of most proteins and nucleic acids. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can bind with proteins weakly and give blue colour.
Glycerol (C3H8O3; mw: 92.09). It is a preservative and a weighing agent. Additon of glycerol (20-30 or 50%) is often recommended for the storage of enzymes. Glycerol maintains the protein solution at very low temperature, without freezing. It also helps to weigh down the sample into the wells without being spread while loading.
Coomassie Brilliant Blue (CBB) (C45H44N3NaO7S2; mw: 825.97). CBB is the most popular protein stain. It is an anionic dye, which binds with proteins non-specifically. The structure of CBB is predominantly non-polar. So is usually used (0.025%) in methanolic solution (40%) and Acetic Acid (7%). Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated in the gel can be removed by destaining with the same solution containing no dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic in nature, it is reported to interfere with staining process. Therefore, large volume of staining solution is recommended. Approximately 10 times the volume of the gel.
Butanol (C4H10O; mw: 74.12). Water saturated butanol is used as an overlay solution on the resolving gel.
Beta Mercapto Ethanol (HS-CH2CH2OH; mw: 78.13). BME was procured from LKB, Bromma, Sweden and was stored at 4°C.
References

1. Anbalagan K (1999). An Introduction to Electrophoresis. Ed. Anbalagan K. pub. The electrophoresis institute, Biotech-Yercaud, Salem, India.
2. Sambrook J and Russel DW (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbour, New York.

SILK dumping- the theory and practice. Is the contingent protection substantiated?

The recent imposition of anti dumping duties on Chinese silk imports to India has stirred up the global silk scenario. The issue whether there is dumping of specific grade raw silks by China is debated with in and out side the countries concerned. This article takes a glimpse at the economic theory behind dumping and anti dumping strategies, as the bulk of the current debates is found to be deficient in the theory behind the phenomenon. The discussion is published in two parts.

Part-1

Dumping is usually understood to mean that a product is exported at a price lower than the price at which the identical or a similar product is sold by the same producers on the exporting country’s domestic market. Dumping can be either a result of monopoly or an instrument to create or strengthen monopoly.
The conditions mandatory for dumping to take place are
1. Presence of an imperfect market where price discrimination between markets is possible. (Because in imperfect markets firms are price setters not price takers) 2. Segmented markets where there is no arbitrage easily possible between markets.
Only if these two conditions are satisfied is it possible for the exporting firm to engage in dumping. For any firm, price discrimination in favor of exports is more common because the share of exports is usually lesser than the domestic demand.
The classic view at dumping identifies the following types
· Private long term dumping and price discrimination
· Subsidized and public dumping
· Dumping in the interest of the exporting country
· Short term dumping
Private long term dumping and price discrimination
Private long-term dumping and price discrimination can result from the profit maximizing policies of a discriminating monopolist. The behavior of the monopolist will be based on the elasticity of demand existing in the domestic and foreign markets. Suppose the domestic demand is less elastic compared to the foreign demand he will restrict the supply of goods in the domestic market (to raise the prices) and dump the product in the foreign market to take advantage of the price elasticity of demand existing there. This concept can be graphically represented.

figure:1


In the figure 1; Dh D’h and Df D’f represent the domestic and foreign demand curves respectively. Mh M’h and Mf M’f represent marginal domestic and marginal foreign revenue curves. The horizontal summing of the two marginal revenue curves gives the aggregate marginal revenue curve Ma M’a. The domestic producers marginal cost curve is C C’. The discriminating monopolist will produce at the point where his aggregate marginal revenue is equal to his marginal cost. Total output will be OR, sales at home- O Wh, and sales abroad- O Wf. The producer has equated his marginal revenue in both markets to his marginal cost, hence satisfying the condition for his private optimum. The price at home is O Ph and price of export is O Pf. In the diagram it is assumed that at the relevant points the elasticity of demand at home is lower than that abroad, so that the domestic price is higher, and thus there is dumping. The essence of private dumping is price discrimination.
Subsidized and public dumping
This is a case of competitive market, not monopoly. The case of an export subsidy is represented in figure 2. The domestic supply curve, which is equal to marginal costs is represented by Sh S’h. Domestic demand curve is Dh D’h. Here price is equal to marginal cost of production. The foreign demand is assumed to be perfectly elastic as represented by the foreign demand curve Df D’f. An export subsidy of Df S is installed which raises the domestic price to OS. Consumption falls and production rises. For the higher domestic price to be possible there have to be either transport costs or a domestic tariff. Domestic consumers pay the price OS and foreign consumers pay a lower O Df. Thus there is dumping.

figure:2
The case of public dumping is more or less the same. Here we are concerned with export marketing arrangements in which there are private competitive producers and a marketing board intervenes to protect exports, rather than maximizing the profit of producers. The board will charge a higher price to the domestic consumer and the profit margin will be used to provide export subsidy.
Dumping in the interest of the exporting country
As distinct from the private interest of the monopolist, dumping can also be in the national interest of the exporting country. National policy in the form of tariffs can make private long term dumping possible. If the foreign demand curve is perfectly elastic then a price equal to the marginal cost can be put for both the domestic and foreign markets. If the foreign demand curve is downward sloping the national optimum demands that in the export market marginal cost to be made equal to marginal revenue and in the domestic market marginal cost to be made equal to average revenue. Thus export price exceeds the domestic price (reverse dumping). In this situation by imposing an optimum export tax the country forces the exporters to bring down the export prices which in effect can lead to dumping.
Short term dumping
Short term dumping is of two types namely sporadic and predatory. Sporadic dumping is the fall in foreign supply prices due to variations in production abroad due to various technical reasons etc. Though a fall in prices is always beneficial to the consumers, it increases the risk of domestic producers.
Predatory dumping is not just a manifestation of monopoly but a technique to maintain it. A foreign dominant supplier may temporarily reduce his supply price so as to force out a domestic producer trying to enter or extent his share of the market, the foreign supplier being able to sustain a price war longer than the domestic supplier. Predatory dumping is effective only if the foreign exporter is able to sustain a monopoly. It is not sufficient for him that he is able to force out a domestic supplier. If he has to compete with other foreign suppliers he may not subsequently be able to raise his price.
Anti Dumping - Meaning and concept
Anti dumping is a measure to rectify the situation arising out of the dumping of goods and its trade distortive effect. Thus the purpose of anti dumping duty is to re establish fair trade. Many countries have anti dumping regulation of some kind. One outcome of the Kennedy round of GATT negotiations was an agreement establishing an international anti dumping code which now governs the anti dumping policies of signatory countries. Dumping refers to private dumping and is considered to occur “if the export price of the product exported from one country to other is less than the comparable price for the like product when destined for consumption on the exporting country. Action could be taken if there is material injury to the domestic producers and sufficient evidence of the injury. Countervailing duties can be imposed to offset subsidies.
The important concern is with short term dumping, which creates uncertainty and may be an instrument of monopoly. The concern is often with producer rather than the consumer interests. If the import price falls there will be domestic income re distribution effect, which should be prevented. Another concern is that of fairness. Dumping seems unfair when it is believed that the export price is below the foreign suppliers average cost of production.
Anti dumping duty and customs duty.
Customs duties fall in the realm of trade and fiscal policies of the Government while anti dumping and anti subsidy measures are there as trade remedial measures. The object of anti dumping and allied duties is to offset the injurious effects of international price discrimination while customs duties have implication for the government revenue and for overall development of the economy. Anti dumping duties are not necessarily in the nature of a tax measure in as much as the authority is empowered to suspend these duties in case of an exporter offering a price undertaking. Thus such measures are not always in the form of duties/ tax. Anti dumping and anti subsidy duties are levied against exporter/ country in as much as they are country specific and exporter specific as against the customs duties which are general and universally applicable to all imports irrespective of the country of origin and the exporter.
Anti dumping (AD) and countervailing (CV) procedures- implications for developing countries
Contrary to their design as temporary means to offset unfair competition, these trade defense measures are in practice used as a long term strategy for various economic difficulties. Used (abused) as a substitute for positive adjustment measures ad and CV actions are also utilized to deal with structural problems. Applied as an instrument for tackling the negative consequences of trade liberalization they became a common tool to protect domestic producers from foreign competition. Faced with the need to protect sensitive domestic industries from increased imports or price slumps, countries often decide to use AD/ CV measures instead of (the more costly) safeguard measures provided for in the GATT 1994.
The WTO era saw a notable rise in AD and CV proceedings. Anti dumping investigations more than doubled and countervailing investigations increased six fold. Countries that appreciated their exchange rate regimes also seem to have used anti dumping to limit current account deficits caused by external shocks.
The almost exclusive restriction of AD initiations to the Big four (Australia, Canada, European Union and United States) was replaced by a broadened field of applicants. Argentina, Brazil, India, Mexico and South Africa became active users, responsible for a significant number of new investigations. Together they account for about one quarter of the AD investigations initiated since 1995. The United States and the European Union initiated two thirds of all CV investigations. Altogether developed countries were behind more than 80% of the overall number for CV investigations initiated in the WTO period.
Anti dumping and countervailing actions has a variety of negative implications.
· They can create substantial distortions with damaging effects on trade and competition.
· The imposition (or even the mere threat) of a duty may lead exporting firms to change production and seek alternative sources of supply.
· The exclusive focus on certain domestic producer neglects costs imposed on consumers due to price increases.
· The existence of these trade defense measures encourages rent seeking behavior by import competing firms.

To be continued.........

Exclusive interview with Dr. Panomir Tzenov, Executive Director of Bulgarian National Center of Agricultural Sciences (NCAS)

Dr. PANOMIR IVANOV TZENOV was born in Vratza, Bulgaria in 1961. He took his MSc from The University of Zootechnics and Veterinary Medicine – Stara Zagora in Animal breeding engineering – sericulture. He obtained his PhD. in sericulture in 1996.
Dr. Tzenov started his career as a technologist in silkworm egg production at the “Silkworm breeding and egg production enterprise”, Vratza, under the Sericulture Experiment Station (SES) Vratza. He became the head of the station in 1987. From 1994 to 2003 he served as The Director of SES, Vratza and as National Director of the project “Rehabilitation of sericulture”, financed by FAO during 2000 – 2002. Presently he holds the position of Executive director of Bulgarian National Center of Agricultural Sciences (NCAS), Sofia.
He is member of various reputed national and international forums such as International Working Group on Sericulture Germplasm” under FAO, International Working group on the global silk handcraft cottage industries and silk enterprises development” under FAO, The Publishing council at the National Center of Agricultural Sciences, Bulgaria etc. He is the founder president of Black, Caspian Seas and Central Asia Silk Association (BACSA).
He has contributed immensely to general sericulture, silkworm breeding, egg production, rearing technology and cocoon and silk processing. He has 164 published papers to his credit and holds author’s certificates for two commercial silkworm hybrids.
Dr. Tzenov is widely traveled and has participated in more than 30 scientific conferences across Asia and Europe.

Inspite of his busy schedule he has found time to give an exclusive interview to 'The Silkworm' for which The Silkworm is thankful.

Tell me about BACSA, its objectives, membership, mode of operation etc.
The Black, Caspian Seas and Central Asia Silk Association (BACSA) was established after the “International Workshop on Revival and Promotion of Sericultural Industries and Small Enterprise Development in the Black, Caspian Seas and Central Asia Region”, organized by FAO in cooperation with the Government of Uzbekistan and held at Tashkent, Uzbekistan from 11 to 15 April 2005 in order to promote sericulture production in the region countries. The association’s main tasks are to: Generate sericulture projects from external resources, including bilateral and multilateral cooperation; Sensitize respective governments and prospective donors; Promote local and regional joint efforts which allow the cooperation between the countries of the Black, Caspian seas region and Central Asia to develop concrete actions that fortify the sustainable development of the sericulture in the region; Promote making agreements for international scientific-technical cooperation and business relations between the countries involved and Promote market studies, training, and dispersion of sericultural germplasm, and silkworm eggs.

For the operation of the association there are, chosen democratically by the members a President, two Vice-presidents, national coordinators for each member country, members of an Executive Committee. The members of the Executive Committee are directly the people in charge of coordination of all the raised activities for their country, within the regional context. The Executive Committee it is the bridge between the country, the national coordinator and the other countries of the association, to execute the actions defined in the region.

The Committee gathers at least once a year and has the following functions: to evaluate the work made by each national coordinator in activities of coordination in her/his country with respect to the BACASA, to recommend the names of the people in the association to receive training abroad. to evaluate and to watch the handling of the "Rotary Funds" and "Research Funds" that will be probably created and to give the recommendations on orientation and better use of these resources, to present/display the research proposals that require financing on the part of the "Research Fund", to approve the necessary resources for this aim and to give recommendations and suggestions on all publications and written material that takes place within the frame of the BACASA and to advise to the association’s President on the advances and progresses that must take place in the development of the activities and give recommendations her/him on the modifications and corrections that are due to make for the final succes of the projects.
Is sericulture a dying enterprise? Does it have a future?
The status of sericulture depends on many factors. In some countries it is really a dying enterprise, due mainly to their high economical development, combined with lack of any governmental support. On the other hand the selling of raw silk/silk allied products by the Chinese at too low prices during the period 1995 – 2005 led to a collapse of the sericulture industry in many countries. The Chinese sold 1 kg of raw silk for US$ 16-18/kg, 3 A quality. That means the fresh cocoon purchasing price should be about US$ 0. 50 – 0.80. Very few countries have so poor farmers who are ready to produce cocoons at this price. On the other hand in many countries the sericulture farmers are supported by the government. In the last year the raw silk price in China jumped to about US$ 43/kg. Therefore the increasing price of the raw silk at the international market, combined with stable state policy to support the sericulture development are the two key factors for the bright future of sericulture. The availability of local silk market is also very important.
Silkworm rearing within the European union countries is considered as one of the protected and promoted activities, being subsidized by Euro 133 per box of 20.000 eggs. This subsidy creates a considerably high income to the farmer, since it approximately duplicates his total income, added to the cocoon value. The replacement of traditional crops by perennial mulberry cultivation is additionally subsidized by the EU by means of the initial installation cost and the income loss for twenty years.
Considering the above I can make the final conclusion that the sericultural industry has a good future.
What is the relevance of sericulture as an agro industry in the developing and underdeveloped countries?
Apart from giving a high-value product, sericulture is a highly women/old people- focused, labour intensive, rural based, income leveler. From one hectare of irrigated mulberry, from the stage of mulberry cultivation to the stage of weaving and marketing, about twelve persons get employment throughout the year. Since the producers of silk cocoons are mostly small farmers while the costly silk final products can be purchased only by the affluent, it transfers income from the rich to the poor, from the urban to rural families. Internationally also, the silk producing countries are mainly the developing countries while silk products have a high demand in the EU countries, Japan and the USA.
Sericulture is also a drought-proffer because, the mulberry plant whose roots go deep in the soil, does not die during prolonged drought and its leaves will sprout whenever there is rain. In the tropical countries, even with 40% rainfall, there will be two crops of mulberry instead of the normal four or five which means, even in the worst drought year, there will be 40% of normal income for the sericulture families. In some temperate /subtropical countries where the summer is very dry and autumn rearing is impossible without irrigation, is possible to get at least one spring cocoon crop without any irrigation of mulberry.
Sericulture thus scores on every point: One of the highest income from one unit of land; Use of less water and drought resistance; Income leveler - transfer of income from rich to the poor nationally and internationally; Generating high rural employment; Gender benign in favor of women-employment; Silkworm rearing is indoors and has no arduous work-drudgery; A natural fibre and ecologically harmless; Hygroscopic, absorbs body moisture and therefore comfortable to wear; No synthetic substitute and highly demanded in EU countries and the USA; No competitor to food crops.
However, in new areas initial promotion and investment is needed in the establishment stage. This is because, while mulberry can be grown without much difficulty, the art and science of silkworm rearing, marketing arrangements for cocoons, reeling and weaving have to be organized in the initial stages till the industry takes root.
The reeled silk yarn is not perishable and being a low-volume, high-value product it has a ready market and can be transported easily. In fact across countries it is air-freighted and not sent by surface transport as transport cost is a small fraction of the total cost. The more difficult task is the teaching of rearing and organizing of reeling to create a market for cocoons produced by the farmer. Once these two aspects are taken care of, sericulture will entrench itself and will develop. Silk being a natural fiber and hygroscopic, it is comfortable to wear in all seasons as it absorbs body moisture. Sericulture thus fits neatly into the economy of any developing country with a significant rural sector, for quite a few decades to come.
Silkworm has long been identified as a laboratory tool (courtesy Dr. Tazima) but yet to be exploited as one. Why is it so?
Recently the silkworm was used as a laboratory tool for the medicine purposes.
The silkworm genome project is underway. What are its implications to the scientific field and agricultural (sericulture) field?
Scientific field: getting new scientific information about the animal genome.
Agricultural field: creation of new silkworm breeds and hybrids combining precious characters, for example sturdiness with high productivity.
While considering the long history of domestication, the bulk of breeding experiments done, viability to genetic manipulation, genomic information, versatility of sericigenous fauna etc., do you think paucity of competent researchers has been the worst handicap for the sector? What are your suggestions for a remedy?
I do not think that paucity of competent researchers has been the worst handicap for the sector. There are many other reasons, the main ones – insufficient financing, considering the sericulture as a minor sector etc.

Dr. Panomir Tzenov can be contacted at panomir@yahoo.com. For more information on BACSA visit http://www.bacsa-silk.org/index.php?lang=bu

Good news for silk

The provisional production figures released by the Central Silk Board (CSB) revealed that domestic raw silk production is showing an upswing with a record of 18760 tons of raw silk during fiscal 2007. The upward move represents 8.41 percent surge over the previous year figures of 17305 tons; however, the record production was much more than the peak production of 17351 tons achieved in 2001-02. During 2002-03 and 2003-04, the drought conditions in the southern region had deterring effect in silk production as farmers went to the extent of even uprooting the mulberry plantations; whereas the ill effects of the severe drought appear to thin out as mulberry silk production had gathered momentum for the fourth successive year.
Last year, the mulberry silk production, including that of bi-voltine silk was 16805 tons up from 15445 tons from the previous year. The redeeming factor of last year's production however was the signs of superior bi-voltine silk galloping in growth by 12.77 percent to cross the 1000 ton mark.
However, the increase in production only proves that bi-voltine silk had come to stay and was finding increased acceptance at farmer's level every year. For more related news visit http://www.bharattextile.com/newsitems

Uzi egg on larval body!

Heat shock proteins – a forgotten link in Silkworm breeding for robustness

Silkworm is one of the most thermal-sensitive organisms. Intensive and careful domestication over centuries has apparently deprived the insect of opportunities to acquire thermo tolerance. Among many factors attributed to poor performance of the bivoltine strains under tropical conditions the major aspect is that many quantitative characters decline sharply when temperature is higher than 28°C. The risk of hybridization of polyvoltine to bivoltine could not be taken due to the delay in fixation of economic characters. The long and hard struggle to evolve robust-productive silkworm hybrids has not so far met with satisfactory results.

The front ranking breeders in the field agrees to the fact that it is a difficult task to breed such bivoltine breeds, which are suitable to high temperature environment and yet productive. Therefore means other than the conventional breeding methods are to be adopted to attain the goal. With the aid of modern biotechnological tools it may be possible to quantify the factors responsible for the expression of temperature tolerance. Resistance to high temperature has been recognized as a heritable character in silkworm and the possibility for temperature tolerant silkworm races were suggested by Kato as early as 1989. Thorough understanding of the phenomenon of temperature tolerance in silkworm is an essential pre requisite for attaining any results in this direction.

Heat Shock Proteins

It is known that rapid heat hardening can be elicited by a brief exposure of cells to sub-lethal high temperature, which in turn provides protection from subsequent and more severe temperature. In 1962, Ritossa reported that heat and the metabolic inhibitor dinitrophenol induced a characteristic pattern of puffing in the chromosomes of Drosophila. This discovery eventually led to the identification of the heat-shock proteins (Hsp) or stress proteins whose expression these puffs represented. Beginning in the mid-1980's, investigators recognized that many Hsps function as molecular chaperones and thus play a critical role in protein folding, intracellular trafficking of proteins, and coping with proteins denatured by heat and other stresses. Accordingly, the study of stress proteins has undergone explosive growth.

Heat-shock proteins are classified into families on the basis of sequence homology and typical molecular weight as Hsp 110, Hsp 100, Hsp 90, Hsp 70, Hsp 40, Hsp 10 and small heat- shock protein families. In eukaryotes many families comprise multiple members that differ in inducibility, intra cellular localisation and function.


Extensive studies have been conducted on the heat- shock response in insects such as Drosophila, Chironomous, Lymantria dispar, the tobacco hornworm-Manduca sexta, the desert ant-Cataglyphis, the fleshfly-Sarcophaga crassipalpis, the locust Locusta migratoria etc. There are reports on the activity of hest shock proteins in silkworm. Evegnev et. al. (1987) studied heat shock response in Bombyx mori cells. Temperature elevation induced active transcription of heat shock mRNAs in infected cells. But at the level of translation headstock treatment failed to induce hsp synthesis and was not able to inhibit production of polyhedrin in such cells.

Joy and Gopinathan in 1995 reported the appearance of 93, 70, 46 and 28 kDa protein bands consequent to high temperature exposure in Bombyx mori. in both bivoltine and multivoltine strains, but with variying kinetics. Lee et.al., in 2003 cloned a genomic DNA fragment containing a promoter region for the gene encoding an HSC70-4 homologue, the structure of which was deduced from the partial cDNA sequences that were registered in a Bombyx mori EST date base. The deduced amino acid sequence with 649 residues was 89% and 96% identical to those from Drosphilla melanogaster hsc-4 and Manduca sexta HSC-70-4 respectively. The expression analysis by reverse transcription PCR demonstrated that mRNA transcription occurred in all tissues examined and was not stimulated by heat shock. Thus HSC70-4, the molecular chaperon is ubiquitously expressed in every tissue of Bombyx mori.

Considering the enormous investigations conducted on HSPs in a plethora of organisms ranging from bacteria to man, it is felt that there is an acute shortage of literature on the heat shock response of the silkworm Bombyx mori. There is dire necessity for 1. Understanding the molecular mechanism of temperature tolerance in silkworm. 2. Identification of the various families of HSPs synthesized and the threshold temperature, which induce their expression. 3. Understanding the differential expression pattern of various HSPs in bivoltine and polyvoltine races and 4. To locate the genes responsible for the heat inducible HSPs and subsequent steps to introgress the same into the bivoltine genome either by conventional breeding or by use of molecular techniques.

The silk factories.......

An experiment on Temperature induced protein synthesis

Differential Expression Of Temperature Induced Protein Synthesis In Two Races of Silkworm Bombyx mori.L

A study was conducted to analyze the differential expression of temperature induced protein synthesis in two silkworm races exhibiting different temperature tolerance. A temperature tolerant race namely Nistari and another temperature non-tolerant race namely CSR2 were used for the study. Experimental animals of both the races were subjected to high temperature treatment at 36°C and 40°C for two different durations, viz., 1 h and 6h on the 2nd day of 3rd, 4th and 5th in star. Proteins purified from the haemolymph collected from the treated larvae immediately after treatment and after four recovery periods (1h, 6h, 24h and 48h) were subjected to SDS PAGE analysis to observe the protein kinetics. The protein profiles of both the races under various treatments when compared with control indicated deviations mostly in the 68-97 kDa region apart from the appearance of new bands. Both the races showed similar protein banding patterns, though the intensity of the bands varied with treatments. Induction of absolutely new bands was observed in the of 97-205, 68-97 and 29-43 kDa molecular weight region possibly representing Hsp: 100, 90, 70 and 34-35kDa. Families. The results are indicative of the differential capability of the races to respond to heat shock by temperature induced protein synthesis. The study results suggest the possibility of using heat shock protein based breeding strategy for inducing robustness in productive breeds.

Hi everybody

I started this blog in honour of the silkworm, a tiny animal belonging to class insecta which produce one of the most sought after materials by man- silk. I wish to cover everything related to this humble creature. Its history- more than 4000 years old; the emperors who adored it- Huang- De of China to Tippu of India, the path it had travelled - the dry deserts of Takli makkan to the snow clad peaks of Himalaya, its biology - of interest to entomologists, microbiologists, pathologists, anatomists, physiologists and molecularbiologists; its economic uses - of interest to women, men the rich and the poor and finally its future possible role on the fate of many countries. Please visit this blog again.

gkrajeshrajesh@gmail.com

The metamorphosis is in progress.......................