Golden Rice:Transgenic Crop

Golden Rice:Transgenic Crop

Introduction

At a New Delhi Biotechnology workshop on February 12, 2001, Professor Ingo Potrykus, Swiss Federal Institute of Technology, Zurich, announced that India will receive technology transfer for the production of Golden Rice, free of encumbrances and license fee.This news brought much cheer into the hearts of many in the country, but most people are blissfully unaware of the stormy seas of controversies in which a handful of nascent seeds of Golden Rice are struggling to survive and to become global giving succor to many a poor.
Golden Rice is an important issue for countries like India.Public awareness of the benefits of this product will help in preventing  repeats of what has been happening with B.T cotton in India.

What is Golden Rice?
Golden Rice is a transgenic variety of rice, with genes for the synthesis of beta-carotene taken from the temperate garden favorite Narcissus pseudo narcissus (daffodil) and inserted into the genome of a temperate strain of rice, using Agrobacterium tumefaciens as the vector, to effect the transfer.The gene construct also contains some genes for enzymes of the bio synthetic pathway of b-carotene, from another bacterium  Erwinia uredovora.The grains of this Genetically Modified (GM) rice  are similar to other varieties, in their crinkly and scabrous husks but the core of the grain is pale yellow, instead of pearly white.The color is due to b-carotene, which makes this the Golden Rice, more so because b-carotene is very important to our health.

The detailed technology used to develop Golden Rice was published by Potrykus and his associates, in Science (vol. 287,  pp 303-305, January, 2000). Ever since Professors Ingo Potrykus  and Peter Beyer (University of Freiburg, Germany) have announced their success in producing Golden Rice, about a year ago, there has been an incessant increasingly polarized public debate over the pros and cons of not just Golden Rice, but all GM crops and organic produce. 
 

The Carotenoid Pool

• About 600 structures of a group of chemical compounds called carotenoids have been discovered in various organisms, but predominantly confined to plants.
• The carotene are hydrocarbons, while the other carotenoids  also contain oxygen.The carotenoids are insoluble in water but are soluble in fats and organic solvents.
•  Several carotenoids occur simultaneously in organisms, and are in a continuous state of metabolic flux. They are referred together as the carotenoid pool, the same way as the amino acid pool  and the nucleotide pool.   
• About 150 of the carotenoids are widely found, among which about 20 closely related carotenoids occur in a wide variety of higher plants,  sea weeds,  animals, fungi and bacteria.    
• Carotenoids are light harvesting pigments located in the plastids.   
• The carotenoids in the chloroplasts protect chlorophyll from damage by excessive light. The carotenoids, related to and derived from b-carotene, impart pale yellow to light reddish yellow colour to the parts in which they occur.   
• Organisms contain different combinations and quantities of carotenoids in different parts.   
• Leaves and other green parts, anthers, pollen, styles and stigmas, fruits and seeds of several species of plants contain carotenoids.   
• Leafy vegetables, mangoes, peaches,  oranges, saffron, maize kernels, yellow-red  coloured pulses, carrots, beetroot, sweet potato, etc., are the common examples.   
• In the earlier days, the techniques of chemical analysis were not sophisticated enough to precisely distinguish one carotenoid from the other, and quantify them.   
• Hence most of the earlier literature collectively called them all carotenoids.   
• HPLC is now routinely used to isolate and quantify different carotenoid compounds.    

  The more important of the carotenoids and their sources are listed below:

1. a-carotene:  leaves and roots of carrots, and the fruit of red palm oil plant 
2. b-carotene:  a very large number of plants and animals, abundant in brown sea
3.                        weeds but exceptionally high concentrations in the daffodils
4. d-carotene:   tomato fruits  
5. g-carotene:   many flowers and fruits, but more abundant in the fungi 
6. Lycopene:    tomato fruit
7. b-cryptoxanthin: maize seeds, eggs, butter, and blood.  
8. Spirilloxanthine: photosynthetic bacteria and some fungi 
9. Lutein (xanthophylls): flowers, fruits, seeds, eggs and animal fats 

As humans and animals cannot synthesize carotenoids, they must be obtained from vegetable dietary sources. 

 
b-Carotene, Vitamin-a and our health

1.   b-carotene is extremely important to us as it is the  most efficient precursor of retinol  (vitamin-A), hence it is called pro-vitamin-A.    
2. The liver converts b-carotene into vitamin-A, the only way we can get our requirement of vitamin-A.  
3. Specific enzymes split one molecule of b-carotene into two molecules of vitamin-A.
4.  Deficiency of vitamin-A causes dry skin, dry eyes, dry mucous surfaces, retarded development and growth, sterility in males and night blindness and other types of irreversible blindness.
5.  Every year, at least a million children die weakened by vitamin-A deficiency and about 3,50,000 others go blind.   Millions of others, young and old, suffer from several disorders related to vitamin-A deficiency (VADs).   

Lycopene 

Lycopene and lutein do not yield vitamin-A.   

a-carotene and b-cryptoxanthin are converted into vitamin-A, but only one molecule of vitamin-A is formed from one molecule of these compounds, while the rest of the molecule adds to the chemical debris of the body.Hence, maize kernels, eggs and animal fats like butter are poor sources of pro-vitamin-A, while tomato is not a source at all.  

 
Disease Prevention By b-Carotene

• In recent times, b-carotene has been rated high as an antioxidant which scavenges free-radicals, which are believed to be involved in the onset of several disorders, including cardio-vascular disease and certain types of cancer
•  b-carotene is being increasingly used as a preventive measure against these diseases.

 
Our Sources Of b-Carotene

• The rice plant produces b-carotene in the green tissues but there is none in the starchy endosperm which constitutes  90 percent of the grain we eat.  
• Most of us get the required amount of b-carotene from supplementary food such as carrots, fruits and leafy vegetables.   
• Several communities, such as the Japanese, consume brown sea weeds, which are high in b-carotene.    
• Throughout the world, the poor do not get enough of b-carotene and hence suffer from vitamin-A deficiency.   
• Vitamin-A deficiency may also be due to certain diseases like measles.  

Why Golden Rice?
About a dozen years ago, Gary Toenniessen, Director of Food Security, Rockefeller Foundation, has recognized the lack of b-carotene in polished rice and identified as a worthwhile goal,  the transfer of genes for b-carotene synthesis into food grains, particularly rice,  using transgenic technology.Such a fine tuned task is beyond the possibilities of traditional plant breeding techniques to achieve. 

• Potrykus saw a hope for millions of poor children in introducing the gene for b-carotene in the rice itself, which is the staple food.   
• The synthesis of b-carotene in plants is a complex process controlled by several genes, which make the task of producing a transgenic much more difficult.  
• Peter Beyer, an expert on the biosynthetic pathway of b-carotene in daffodils, became an associate.   
• The choice of daffodils as the source of b-carotene genes is a very clever one, as they have an exceptionally high concentration of b-carotene, all of which can be converted into vitamin-A, but unfortunately no part of daffodils is edible.

Synthesis of Golden Rice

Potrykus and Beyer faced several difficulties in creating Golden Rice, the more important being:  
a) The transfer of a set of several genes together proved to be a formidable task;  
b)  Rice plants did  not grow well in green houses; and  
c) The genes they transferred and the bacteria used for such transfer were encumbered 
    by patents and proprietary rights. 
 
Finally all the difficulties were overcome.The success was achieved, more importantly,  without industrial financial support.The owners of different patents, Syngenta, Monsanto and four other companies, recently agreed to donate the technology free to developing countries, in exchange for commercial marketing rights in the US and other affluent markets.   The concession offered by the patent owners is the basis for the announcement by Potrykus, at New Delhi,  on a free transfer of technology to India.An appreciable gesture,  but sneered at by the Rural Advancement Foundation International, as “a rip-off of the public trust”.  

Challenges Ahead
A decade’s dream of Potrykus has been realised, but a lot more is yet to be done, the following being the more important challenges:
  a) the gene set has to be transferred to a tropical variety of rice since the temperate
    variety will not fare well  nor will it be accepted, in the tropics;
  b) years’ of lab and field work on perfecting a viable Golden Rice varieties suitable
    for  different agro-climatic zones of India, need to be developed; 
  c) currently the quantity of b-carotene in the golden rice is not good enough to
    provide a substantial quantity of it, at the normal 300g of rice eaten per
    day; 
  d) the product should be affordable to the very poor of the world, for which it should
    get into the Public Distribution System,  and 
  e) the hostile, sometimes violent,   protestors have to be satisfied on the safety (some
   gene products may cause allergies, some GM crops are feared to become    gregarious weeds) and utility of the product (the ultimate desired  benefit may not be available in the end).  

Do GM Crops Become Threatening Weeds?

Amidst the disturbing controversy, an article in the journal Nature (February 8, 2001) came as a whiff of fresh air.   A ten year study in UK by Michael Crawley and team  has shown that GM crops hardier than rice, such as maize, beet, oilseed rape and   potato, did not become more competitive than their non-GM counterparts and did not invade the environment .Rice is a very delicate plant. Golden Rice becoming gregarious is far beyond the realm of possibilities.

Fool’s Gold?
The Greenpeace organization, which is at the forefront of criticism of biotechnology products, posted a statement on the internet on February 9, 2001, and dubbed Golden Rice as ‘Fool’s Gold’.   The criticism is based on the following points:
  a) The promoters of Golden Rice and supporters of GM crops have gone too far in over selling the product as for example by such statements as Syngenta’s projection that a single month’s marketing delay of Golden Rice will cause 50,000 children to go blind;
  b) An adult would have to eat 3.7kg of raw  (around 9kg of cooked) Golden Rice, as
      against the normal intake of 300g of raw rice a day,  to get the daily requirement 
      of b-carotene;  and    
  c)The underlying causes of vitamin-A deficiency (poverty and lack of access to more
      diverse diet) were not addressed and short term measures such as supplementation    
      (e.g., pills) and food fortification which are cheap and effective were not considered.
 

• At a Press Conference on February 9, 2001, at Lyon, France, Ingo Potrykus conceded that the quantity of b-carotene available in the current sample of Golden Rice is small and needs to be improved.   
• He pointed out that it was never meant that Golden Rice alone would supply the entire daily requirement.   
• He assured that golden rice does not compete with alternative and traditional interventions but rather complements them.  

•   With the possibility of wheat, cassava, sweet potato, banana, etc.,  going gold in the future, alternatives to Golden Rice will be several.   
• The future crops will also address other deficiencies such as iron, essential amino acids, etc. 
• Some Points To Ponder Amidst these emotionally charged, and seemingly uncompromising positions of the protagonists  and antagonists of GM crops, we should keep in mind certain issues, particularly in the context of Golden Rice.  
• The concept of balance diet has come into practice as people have realised a very long time ago that no single (or even a few) food items can provide all the nutrients we need.     Potrykus says that Goldeb Rice can now provide 20-40 per cent of the daily requirement.   
• Those who eat Golden Rice would get at least some b-carotene, which certainly will compensate marginal deficiencies.  
• There is no dearth for sources of b-carotene in most countries.     
• There are about 160 species of plants in India, used as food and/or medicine, which contain very considerable amounts of b-carotene in their carotenoid pool.    
• Greenpeace suggested supplementation through pills.   
• As purified b-carotene is expensive costing  about US $ 185/25mg, administration of the recommended dose (5mg/day) through pills  is not feasible.  
• Greenpeace suggested red palm oil as a source of pro-vitamin-A, but what this contains abundantly is a-carotene, which is only half as efficient as b-carotene in terms of conversion into vitamin-A.  
• Besides, palm oil contains about 48 per cent of palmitic acid, a saturated fatty acid, which is not so good for our health.   Then, how much of palm oil one can consume?   How much of it actually is available to the poor who cannot afford even the more commonly available leafy vegetables?
• Vitamin-A requires a (mostly endogenous) protein and palmitic acid to be stable and functional in our body.  
• It also requires tocopherol (vitamin-E), an antioxidant that makes it more stable.   
• When these substances are not present in the existing varieties of rice and other cereals, it is very unreasonable to expect Golden Rice to contain them.  
• May be if given a chance, the future varieties of Golden Rice may contain these stabilising compounds.   
• Their absence does not make Golden Rice a useless product.
• Golden Rice is now only a research product and it takes a lot of time and effort to make it available across the counter, particularly in the developing countries,  which essentially require it.   
• Controversial issues can be settled only through relevant experimental data and discussion

Benefits of Golden Rice  

• Benefits of Golden Rice at the poorest of the poor who cannot get anything other than rice, green chillies and salt, if at all.   
• Traditional wisdom taught all communities to eat adequate quantities of pulses, green vegetables and fruits.   
• But affordability, not availability,  is the problem.   
• Even when Golden Rice is on the market, affordability will be the spoiler, though free technology transfer is a blessing.  
• Even those who can afford supplementary foods do not often get adequate quantities of b-carotene, for various individual metabolic deficiencies or merely out of ignorance.  
• Just as the compulsory use iodised salt, even by those who do not need it, has been introduced so that those needing iodine would get it, Golden Rice too will provide for a compulsory intake of some quantity of b-carotene.  
•  This is to save millions of children in the developing world, from blindness and death

High Security for Biotechnology?
In a civilised society hooliganism and vandalism have no place.   
Destroying or burning GM trial plots, vandalising research laboratories are abominable criminal acts, which transgress the intellectual and material property rights of others.     
Discussion and not destruction is the need of the hour. 
The degree of scare implanted by  biotech antagonists on the minds of biotechnology researchers, is reflected by the fact that Golden Rice seeds are protected in a grenade proof bunker in Switzerland!

Golden Rice Deserves A Chance

• Golden rice and other GM crops are meant to revolutionize agriculture.
• Golden Rice signifies a shift in the target of GM crops, from the farmer to the consumer.     
•  It has the potential to help millions throughout the developing world.   
• Millions of poor in the developing world need it.    
• Golden Rice deserves to be given a chance to prove itself or to the contrary.   Let us hope that the dust settles soon and the sun shines on Golden Rice.

Agrobacterium Mediated Gene Transfer- Genetic Engineering

 Agrobacterium Mediated Gene Transfer- Genetic Engineering

Agrobacterium mediated gene transfer is one of the most widely used methods of gene transfer. Agrobacterium tumefaciens is a bacterial plant pathogen found in the soil which causes crown gall disease in plants.The basic mechanism that works behind it is, transfer of a segment or small piece of bacterial DNA to the plant genome. This bacterial DNA segment gets incorporated in the plant genome, stabilizes there and transcribes or expresses itself.The bacterial DNA causing disease is situated on a plasmid, Ti plasmid (Tumour inducing plasmid, 200kb) instead of on a bacterial chromosome.
The segment which is actually transferred to plant genome is situated on T-DNA (transferred DNA, 23kb) of this plasmid. 
In the expression of disease symptom, two major sets of genes are involved

•     Genes on T-DNA (total 15 genes)
•     Virulence genes i.e. vir genes

Both of the above sets of gene can be present on the same plasmid (co-integrative vectors) or separate plasmid (binary vector).
1.Genes on T-DNA
The T-DNA contains two types of genes, which possess the ability to express in plants:

1.  Oncogenes – Encode for synthesis of auxins and cytokinins (phytohormones). The overproduction of phyto-hormones leads to proliferation of callus or tumour formation. 
2. Opine synthesizing genes – Encode for synthesis of opines (a product from amino acids + sugars, which are produced and excreted by the crown gall infected cells and consumed by A. tumefaciens as carbon and nitrogen sources this means opines act as source of nutrient for bacteria

 
 2.Virulence Genes 
These are located on vir region (30 kb) of Ti plasmid.These genes are not transferred to the plant genome, just help in the process; organized in six operons that are essential for the transfer (virA, virB, virD, and virG) or increase transfer efficiency (virC and virE) of T-DNA.They are involved in activities like recognition of host plant, the chemotaxis, attachment to the plant cell, transfer of DNA in plant cell nucleus and integration of inserted DNA into the plant chromosome.
Procedure :
 For transformation oncogenic T-DNA is removed and replaced by another desired DNA segment with border sequence.While working binary vectors the vir genes are removed and the desired DNA is integrated between the borders.The vir genes necessary for transfer to the plant genome are arranged on a second plasmid with T-DNA and both borders removed.Such binary vectors restrict the transferred Ti plasmid from spreading uncontrollably as genes responsible for that are do not get transferred.
Advantages of Agrobacterium mediated gene transfer 

•     Simple and comparatively less expensive
•     High transformation efficiency
•     Transgenic crop obtained have better fertility percentage
•     Today, protocols for both monocotyledons and dicotyledons are available
•     Relatively large length chromosomal segment can be transferred with little arrangement

Disadvantages of Agrobacterium mediated gene transfer 

•     Time consuming
•     Not all kind of cells can be treated by this method
•     Can not transform organelles
•     Sometimes leads to false positive results

    Genetic engineering Techniques
    Gene Transfer
    Agrobacterium – Gene Transfer

Replication in Prokaryotes and Eukaryotes

Replication

• One of the single strands of the double-stranded DNA molecules is the template for the generation of the other.

• DNA replication is semi-conservative. Each of the two molecules double-stranded molecules resulting from replication contains one old and one new single strand.

Proving How the DNA is Replicated

• Watson and Crick proposed the hypothesis of semi-conservative replication in their original Naturepaper.
• The Meselson Stahl experiment was a real classic in genetic research.
• It was a critical experiment by design. This means that no matter what the result, they would have their answer.

Meselson and Stahl Experiment

• E. coli are grown in heavy nitrogen (¹⁵N) for many generations.
• This caused the nitrogen in the DNA molecule of each cell to contain ¹⁵N, a heavier than typical isotope.
• The E. coli were then grown for one or two cell divisions in ¹⁴N, the lighter and typical isotope.
• DNA was spun in a cesium chloride gradient. Meselson and Stahl actually invented this technique, called density centrifugation, which now has many other applications, just for the purposes of this experiment.
• The cesium chloride gradient and centrifugation separates molecules based on their density.
  • The DNA molecules with ¹⁵N are more dense than those with ¹⁴N, and band below DNA with¹⁴N.
  • If two bands were observed after one division in ¹⁴N, there would have been wholly old strands and wholly new strands. This would have been consistent with and meant the replication was conservative.
  • If there was just one band after one division, replication could be either dispersive or semiconservative.
• The result was just one band after one division.
  • If one or a long smear was observed after two divisions in ¹⁴N containing medium, dispersive replication would have been the mode.
  • If intermediate weight and light weight molecules were found, semiconcervative would be the mode.
• This is what was found; the replication was semiconservative. This was the predicted outcome of Watson and Crick.

DNA Replication: A Closer Look

• DNA polymerase enzymes are involved in repair and chromosome replication.
  • Prokaryotic DNA I: Discovered by the famous Dr. Authur Kornberg - this enzyme polymerizes small DNA segments during replication and repair. Most of the DNA polymerase found in prokaryotic cells is DNA polymerase I.
  • DNA III: This is the primary DNA polymerase involved in DNA replication of bacterial chromosomes.
• DNA always replicates from 5' to 3' in direction using the anti parallel strand as a template.
  • Therefore, for each double-strand of DNA, one strand must replicate in the opposite direction of the other.
  • Why only in the 5' to 3' direction? Because a new base can only be added to the 3' OH position.
  
• Also, a new base can only be added to an existing polymer!
  • So how does it start? (This question is answered later in this unit.)
  • A special DNA template dependent RNA polymerase called primase makes a stretch of RNA that complements the template strand and forms a RNA / DNA duplex.
  • Next, DNA polymerase adds to that RNA molecule.
  • Later, the RNA molecule is removed.

DNA Replication Steps

• Initiation: this involves the assembly of a replication fork (bubble) at an origin of replication sequence of DNA found at a specific site of the circular chromosome of a bacterium. The fork is generated by a complex of proteins called a primosome.
• Elongation: this is the addition of bases by another complex of proteins called the replisome. Parental strands unwind and daughter strands are synthesized.
• Termination: the duplicated chromosomes separate from each other. This is actually a complicated procedure, the details of which we won't cover in this course.

       

DNA polymerase

• Some types of DNA polymerase are occasionally called DNA replicases.
• Other types are considered DNA repair enzymes.
• All DNA polymerases polymerize a polynucleotide by adding to an existing double-stranded stretch of DNA.
• Aggregates of specific subunits of polypeptides makeup the replicase molecule.
• In E. coli, there are three major DNA polymerases: DNA polymerase I, II and III.
• DNA poly I is found in the highest concentration of all DNA polymerases; it is involved in DNA repair and assists with primary DNA replication.

• DNA poly II is exclusively involved in repair.

        

• DNA poly III is the major DNA polymerase role.

• All DNA polymerases add to the 3’ OH of the existing polynuceotide.
• DNA polymerases have other enzymatic activities as well.
  • One is an exonuclease activity, the digestion of the polymer into monomers, which is the reverse activity of polymerization.
  • One can diagram it to look a bit like PackMan of the video game. If the enzyme has exonuclease activity, it works in the 3' to 5' direction, which is the opposite of the 5' to 3' polymerase activity. Some DNA polymerase holoenzyme complexes have both a 3' to 5' and a 5' to 3' exonuclease activity. DNA polymerase I does for example.
  • This function is involved with error reduction: "proof reading". It is also involved with removal of the RNA primers and replacement of the RNA with DNA. There is also a lot of involvement of these activities in non-replicative DNA repair.
  • Another enzymatic activity associated with DNA polymerase is proofreading. This is a check that the correct base was incorporated immediately after the fact.

Fidelity of Replication

• The accuracy or fidelity of DNA replication can be measured relatively easily and has been for many different types of organisms.
• It can be expressed in a number of different ways, for example, errors per nucleotide incorporated.
• It varies substantially between prokaryotes, eukaryotes, unicellular and multicellular organisms.
• A typical value given by some genetics textbooks is 1 error/genome/1000 bacterial replications.
• It would be 10^-3 instead of 10^-8 to 10^-10, if there were no checks on the system.
• Checks include a pre-synthesis check to verify the incoming base is correct.
• The proofreading check verifies that the correct base was incorporated.
• The 3’ to 5’ exonuclease activity assists when it is found that the base incorporated was incorrect. It removes the incorrect base so that the polymerase can try again to get it right.
• The DNA polymerase I enzyme is 103 kD.
• Of this a 68 kD portion is called the Klenow fragment.
• Klenow has the 3’ to 5’ exonuclease and the polymerase activity in different regions of the fragment.
• Another 35 kD fragment has a 5’ to 3’ exonuclease activity.
• The E. coli DNA polymerase I incorporates about 10 bases in length at a time. This is called its processivity. Some organisms poly I incorporate more, some less.

Nick Translation

• Nick translation is an extremely useful technique for labeling DNA.
• First DNA is nicked using a shearing force such as forcing the solution containing the DNA molecules through a fine bore hypodermic needle.
• The DNA with nicks, DNA polymerase I and labeled deoxiribonucleoside triphosphates are combined in solution, and the DNA polymerase I molecules attach to the nick, use their 5’ to 3’ exonuclease activity to digest out existing nucleotides, while replacing them with labeled ones using their polymerase activity.
• This technique can be used to incorporate radioactive labeled nucs.

DNA poly III

• DNA polymerase III is the main polymerase for E. coli chromosome replication.
• It is made up of many subunits.
• It has several different activities.
• It is found in different forms ( subunit associations).
• E. coli's has 3’ to 5’ exonuclease proofreading.

  

• The gene dna E codes for the 130 kD fragment which has the polymerase activity.
• The alpha subunit works to increase error proofreading ability.

Types of Mistakes Made

• Frameshift(s) are extra or missing base(s).
  • Frameshifts are influenced by the processivity of the enzyme.
  • Slippage is less if processivity is greater (adding more bases before disassociating).
  • Strings of some bases, especially short repeats, can increase the frequency of frameshift mistakes.
• Substitutions are when the wrong nucleotide is incorporated.
• Substitution rates are strongly influenced by the proofreading function of the DNA polymerase.

Eukaryotic DNA Polymerase

• DNA Polymerase alpha and delta replicate the DNA.
• DNA polymerase alpha is associated with initiation, and delta extends the nascent strands.

      

• DNA polymerase epsilon and beta are used for repair.
• DNA polymerase gamma is for replication of mitochrondrial DNA.

Priming

• All DNA polymerases require a 3’ end of an existing double-stranded molecule to add nucleotides to.
• There are several different ways this is done.
  • Cells make RNA primers.
  • Some viruses use a preformed RNA.
  • A nick or gap in the phosphodiester backbone of the DNA molecule is used as the starting point.

Semidiscontinuous synthesis

• The replication fork moves in one direction.
• However, DNA is replicated in the 5’ to 3’ direction only.
• How can this be?
• There is a leading and lagging strand.
• Short pieces of DNA called Okazaki fragments with a short stretch of RNA at each 5' end are found temporarily on the lagging strand. 
• (The leading strand might at times appear to be fragmented also, but this is due to deoxi-uracil getting mistakenly incorporated instead of thymine, and then the uracil getting cut out by uracil glycosidase and AP endonuclease.)
• This system of replication is called semi-discontinuous because one strand, the leading, is continuous, and the other, the lagging, is discontinuous.
• Each Okazaki fragment starts with about 10 bases of RNA primer.
• What happens to Okazaki fragments? Does chromosomal DNA really contain RNA also?
• Ligase fails to seal gaps if the gap is between an RNA and a DNA base, which is how the gap exists between each separate Okazaki fragment.
• Time and the gap give DNA polymerase the opportunity to do the Pack Man routine.
  • DNA polymerase I eats the RNA bases (exonuclease activity), whilst incorporating new DNA bases (DNA polymerase activity).
  • Actually, a DNA polymerase I molecule will digest and add a few bases, and then fall off.
  • Because a gap still exists, another DNA polymerase I molecule will do the same again.
  • If the gap is now between two deoxiribonucleotides, either DNA polymerase I or ligase will associate with the gap.
  • If ligase gets their first, it seals the gap.
  • Ligase will "get their first" sooner or later, depending on concentrations, and the gap will get sealed.
• DNA polymerase I is using the end of the last Okazaki fragment, which is composed of DNA, as its primer.
• DNA polymerase I removes and adds bases beyond the RNA primer stretch a bit, unnecessarily.
• Ligase seals the gap.
• Ligases use energy from NADH (E. coli) or ATP (T4 phage).

Primosome

• First, helicase separates the strands of the double-stranded DNA molecule; E. coli's DnaB codes for helicase.
• Single-strand DNA binding proteins maintain the strands in this region as single-stranded.
• This occurs at the oriC site, the bacterial origin of replication.
• Primase, the DNA template dependent, RNA polymerase, which makes the primers, is coded for by the dna G gene.
• Unwinding the DNA by helicase requires ATP as an energy source and substrate.
• A note on unwinding enzymes:
  • Topoisomerases effect the coiling of double-stranded DNA.
  • Type 1 work by making single-strand breaks; Type 2 work by making double-strand breaks.

Stopping

• The replication fork moves in opposite directions away from the origin of replication, forming two bubbles. 
• How does it know when to stop in bacteria, so that just one round of replication takes place, and you don't just get a jumble of many copies?
• In E. coli, there are ter elements, which are 23 base pair consensus sequences.
• These are binding sites for the tus gene protein.
• This stops the Dna B product, helicase, from unwinding DNA in the ter element regions.
• Strands synthesize just up to ter.
• The strand extension is prevented in one direction but can read through in the other direction until the bubbles meet. The two strands must then disconnect from each other.

Summary

• Only one strand can replicate continuously. The other is discontinuous.
• On the discontinuous strand, short segments are called Okazaki Fragments, these are about 1,500 bases in length in prokaryotes, and 150 bases in eukaryotes.
• The continuous is called the leading strand, the discontinuous is the lagging strand.
• The leading has high processivity, it continues until the entire strand is replicated.
• The lagging requires these repeated steps:
  • 1. Primer synthesis
  • 2. Elongation
  • 3. Primer removal
  • 4. Ligation (joining the pieces). 
• Where as DNA Poly III is involved in the leading strand synthesis and adding bases to the primers of the lagging strand, DNA Poly I later recognizes the RNA primers and removes them with its exonuclease activity and replaces the RNA with DNA.
• Subsequently, patches must be made. Because connection of the separately synthesized fragments do not involve incorporation of a nucleoside triphosphate, a special enzyme does this,Lygase.to put it all together.

Origins of Replication and Replication in Eukaryotes

• The DNA sequences required as origins of replication in eukaryotic cells are called Autonomously Replicating Sequences (ARS).
• They contain an eleven base pair core consensus sequence.
• Progress has been made toward the replication of the DNA of eukaryotics in test tubes using eukaryotic enzymes.
• Two different DNA polymerases are required.
• One for the leading and another for the lagging strand.
  • In prokaryotic cells rates of replication are 500 bases per second.
  • In eukaryotic cells rates are 50 bases per second. Eukaryotes have 100 to 3,000 times more DNA than prokaryotes.
  • Bacteria have only one ARS, but eukaryotes have many.
  • In eukaryotes, clusters of origins in certain regions of the chromosome are active at a given time.
  • Each origin is separated by 30,000 to 300,000 base pairs.
  • There is a repeatable order to the activation of each region of the chromosome.
  • Early to replicate regions include areas of euchromatin.
  • Late to replicate regions are heterochromatin.
  • There is a yet unknown mechanism that blocks replication from occurring twice in the same division cycle.

Histone Synthesis and Assembly

• Histones are coded for by multiple copies of the coding sequence.
• They are produced during S Phase, and not during other times of the cell cycle.
• The amount of histone protein is equal to the total mass of chromosomal DNA.
• The mRNAs breakdown rapidly after S Phase.
• How can different types of histones associate with the correct regions of DNA?
• Old come back together in the same region as they were prior to replication, and the old ones influence the modifications made to the new ones in the same area.
• Assembly occurs just after strand synthesis but the nucleosomes mature for an hour after synthesis.

Telomeres

• These are the short G-rich repeats found at the end of the linear chromosomes of eukaryotes.
• The repeated sequence of GGGTTA make up the human teleomeres.
• Telomerase is the enzyme that makes these.
• Telomerase has a bound RNA template that it uses over and over to create these repeats.
• It is a reverse transcriptase.