Synthetic Chromosome

'Synthetic' Chromosome Permits Rapid, On-Demand 'Evolution' of Yeast; Artificial System Has Built-In Diversity Generator

In the quest to understand genomes -- how they're built, how they're organized and what makes them work -- a team of Johns Hopkins researchers has engineered from scratch a computer-designed yeast chromosome and incorporated into their creation a new system that lets scientists intentionally rearrange the yeast's genetic material.

A report of their work appears September 14 as an Advance Online Publication in the journal Nature.

"We have created a research tool that not only lets us learn more about yeast biology and genome biology, but also holds out the possibility of someday designing genomes for specific purposes, like making new vaccines or medications," says Jef D. Boeke, Ph.D., Sc.D., professor of molecular biology and genetics, and director of the High Throughput Biology Center at the Johns Hopkins University School of Medicine.

Boeke notes that yeast is probably the best-studied organism with a nucleus on the planet and is "already used for everything from medicine to biofuel," making it a good candidate for his team's focus.

In designing the synthetic yeast chromosome, Boeke says, the goal was to make it maximally useful to researchers by laying down some ground rules: First, the product could not compromise yeast survival; second, it must be as streamlined as possible; and third, it had to contain the capacity for genetic flexibility and change.

Using the already known full genetic code -- or DNA sequences -- of the yeast genome as a starting point, Johns Hopkins graduate student Sarah Richardson wrote a software program for making a series of systematic changes to the DNA sequence. The changes were planned to subtly change the code and remove some of the repetitive and less used regions of DNA between genes, and to generate a mutated "version 2.0" of a yeast cell's original 9R chromosome. The smallest chromosome arm in the yeast genome, 9R contains about 100,000 base pairs of DNA and represents about one percent of the single-celled organism's genome.

Building the actual chromosome started with stringing individual bases of DNA together that were then assembled into longer segments. Large segments of about 10,000 base pairs were finally put into live yeast cells and essentially swapped for the native counterpart in the chromosome, a process for which yeast are naturally adept. In addition to 9R, the team also made a smaller piece of the chromosome 6L. Yeast cells containing the synthetic chromosomes were tested for their ability to grow on different nutrients and in different conditions, and in each case came out indistinguishable from natural yeast.

The Hopkins teams says what distinguishes this constructed chromosome from the native version -- and sets it apart from other synthetic genome projects -- is an "inducible evolution system" called SCRaMbLE, short for Synthetic Chromosome Rearrangement and Modification by Lox-P mediated Evolution.

"We developed SCRaMbLE to enable us to pull a mutation trigger -- essentially causing the synthetic chromosome to rearrange itself and introducing changes similar to what might happen during evolution, but without the long wait," explains Boeke. Why build in the scrambling system? To change multiple things at once, says Boeke, which is anathema among experimental scientists who traditionally change only one variable at a time, Nature is never that well controlled, he says.

The team activated SCRaMbLE in yeast containing both the synthetic 9R and 6L chromosomes, then analyzed the DNA from the yeast cells. Testing this population of SCRaMbLEd yeast fed various nutrients they found some grew fast, some grew slowly and others really slowly, and some of the fast-growing ones had very specific defects resulting from specific gene loss, showing that SCRaMbLE does indeed introduce random variation. When the team analyzed the molecular structure of the synthetic 9R and 6L chromosomes from this SCRaMbLEd population, they found chromosomes with small deletions, rearrangements, and other alterations, at wildly varying locations.

"If you think of the yeast genome as a deck of cards, we now have a system by which we can shuffle it and/or remove different combinations of 5000 of those cards to get lots of different decks from the same starter deck," Boeke says. "While one derivative deck might yield good hands for poker, another might be better suited for pinochle. By shuffling the DNA according to our specifications, we hope to be able to custom design organisms that perhaps will grow better in adverse environments, or maybe make one percent more ethanol than native yeast."

Boeke says the 9R and 6L experiments are "the beginning of a big project, whose ultimate goal is to synthesize the whole yeast genome (about 6000 genes) and SCRaMbLE the 5000 likely to be individually dispensable. And he wants to make the tool available to anyone who wants to use it, without intellectual property protection.

Major support for this study came from the National Science Foundation, with other contributions from Microsoft, Department of Energy, and Fondation pour la Recherche Médicale.

In addition to Boeke, Johns Hopkins scientists who contributed to the Nature study are Jessica S. Dymond, Sarah M. Richardson, Candice E. Coombes, Timothy Babatz, Joy Wu Schwerzmann, Héloïse Müller, Narayana Annaluru, Annabel C. Boeke, Junbiao Dai, Srinivasan Chandrasegaran, and Joel S. Bader.

Also, William J. Blake of Codon Devices; and Derek L. Lindstrom, and Daniel E. Gottschling of Fred Hutchinson Cancer Research Center.

Blood Mystery Solved: Two New Blood Types Identified

Two New Blood Types Identified

You probably know your blood type: A, B, AB or O. You may even know if you're Rhesus positive or negative. But how about the Langereis blood type? Or the Junior blood type? Positive or negative? Most people have never even heard of these.

Yet this knowledge could be "a matter of life and death," says University of Vermont biologist Bryan Ballif.

While blood transfusion problems due to Langereis and Junior blood types are rare worldwide, several ethnic populations are at risk, Ballif notes. "More than 50,000 Japanese are thought to be Junior negative and may encounter blood transfusion problems or mother-fetus incompatibility," he writes.

But the molecular basis of these two blood types has remained a mystery -- until now.

In the February issue of Nature Genetics, Ballif and his colleagues report on their discovery of two proteins on red blood cells responsible for these lesser-known blood types.

Ballif identified the two molecules as specialized transport proteins named ABCB6 and ABCG2.

"Only 30 proteins have previously been identified as responsible for a basic blood type," Ballif notes, "but the count now reaches 32."

The last new blood group proteins to be discovered were nearly a decade ago, Ballif says, "so it's pretty remarkable to have two identified this year."

Both of the newly identified proteins are also associated with anticancer drug resistance, so the findings may also have implications for improved treatment of breast and other cancers.

As part of the international effort, Ballif, assistant professor in the biology department, used a mass spectrometer at UVM funded by the Vermont Genetics Network. With this machine, he analyzed proteins purified by his longtime collaborator, Lionel Arnaud at the French National Institute for Blood Transfusion in Paris, France.

Ballif and Arnaud, in turn, relied on antibodies to Langereis and Junior blood antigens developed by Yoshihiko Tani at the Japanese Red Cross Osaka Blood Center and Toru Miyasaki at the Japanese Red Cross Hokkaido Blood Center.

After the protein identification in Vermont, the work returned to France. There Arnaud and his team conducted cellular and genetic tests confirming that these proteins were responsible for the Langereis and Junior blood types. "He was able to test the gene sequence," Ballif says, "and, sure enough, we found mutations in this particular gene for all the people in our sample who have these problems."

Transfusion Troubles

Beyond the ABO blood type and the Rhesus (Rh) blood type, the International Blood Transfusion Society recognizes twenty-eight additional blood types with names like Duffy, Kidd, Diego and Lutheran. But Langereis and Junior have not been on this list. Although the antigens for the Junior and Langereis (or Lan) blood types were identified decades ago in pregnant women having difficulties carrying babies with incompatible blood types, the genetic basis of these antigens has been unknown until now.

Therefore, "very few people learn if they are Langereis or Junior positive or negative," Ballif says.

"Transfusion support of individuals with an anti-Lan antibody is highly challenging," the research team wrote in Nature Genetics, "partly because of the scarcity of compatible blood donors but mainly because of the lack of reliable reagents for blood screening." And Junior-negative blood donors are extremely rare too. That may soon change.

With the findings from this new research, health care professionals will now be able to more rapidly and confidently screen for these novel blood group proteins, Ballif wrote in a recent news article. "This will leave them better prepared to have blood ready when blood transfusions or other tissue donations are required," he notes.

"Now that we know these proteins, it will become a routine test," he says.

A better match

This science may be especially important to organ transplant patients. "As we get better and better at transplants, we do everything we can to make a good match," Ballif says. But sometimes a tissue or organ transplant, that looked like a good match, doesn't work -- and the donated tissue is rejected, which can lead to many problems or death.

"We don't always know why there is rejection," Ballif says, "but it may have to do with these proteins."

The rejection of donated tissue or blood is caused by the way the immune system distinguishes self from not-self. "If our own blood cells don't have these proteins, they're not familiar to our immune system," Ballif says, so the new blood doesn't "look like self" to the complex cellular defenses of the immune system. "They'll develop antibodies against it," Ballif says, and try to kill off the perceived invaders. In short, the body starts to attack itself.

"Then you may be out of luck," says Ballif, who notes that in addition to certain Japanese populations, European Gypsies are also at higher risk for not carrying the Langereis and Junior blood type proteins.

"There are people in the United States who have these challenges too," he says, "but it's more rare."

Other Proteins

Ballif and his international colleagues are not done with their search. "We're following up on more unknown blood types," he says. "There are probably on the order of 10 to 15 more of these unknown blood type systems -- where we know there is a problem but we don't know what the protein is that is causing the problem."

Although these other blood systems are very rare, "if you're that one individual, and you need a transfusion," Ballif says, "there's nothing more important for you to know."

Excellence and Relevance for Human Kind

Biotechnology - In Pursuit of Excellence and Relevance for Human Kind

The Department of Biotechnology is committed to generate excellence in biotechnology research for a strong, indigenous knowledge base and to ensure its application as a premier precision tool for creation of economic wealth for the welfare of the society and national prosperity. The last four decades represent the age of new Biology. Today biological sciences have become the harbinger of new and hitherto unimagined products and processes in the areas of food, feed, health, animal and plant productivity, environment conservation and energy generation. Sustainable development of our resources for ensuring food, nutritional, health, environmental and livelihood security of the people by harnessing the powers of biotechnology is a dream of the scientific community, being translated into products, processes and technologies. The focus during these two years has been on basic and application oriented new biology and biotechnology covering agriculture and plants, genomics, molecular medicine, bioinstrumentation, bioinformatics, biofuels, biofertilizers, biopesticides, human resource development and environment and biodiversity. The Department has developed biosafety protocols and guidelines related to ethical issues. Partnership with the industry for commercialization of technology and marketing of bioproducts is another area where there has been some success. A major pursuit thus has been to bring synergy between researchers, academicians, industry, policy makers and public. Vision - Biotechnology -A Ten Year Perspective On September 7, 2001, Hon'ble Prime Minister, Shri Atal Bihari Vajpayee released the 'Biotechnology - A Vision - Ten Year Perspective' document of the department. The expert advice from a large number of scientists from India and abroad has been obtained in its preparation. The goals outlined in the vision document are achievable within ten years for a biotechnology based, environmentally sound, sustainable societal development and a bioindustrial revolution through excellence in bioscience innovations, discoveries and increased understanding of life processes.  Our vision is 'Attaining new heights in biotechnology research, shaping biotechnology into a premier precision tool of the future for creation of wealth and ensuring social justice – specially for the welfare of the poor'. Areas where significant progress has been made with important achievements are briefly touched upon as below : Crop Biotechnology : Successful transgenic research has been carried out on banana, cabbage, mustard, mungbean, and wheat for crop improvement, increased nutritional value, disease resistance, and better shelf life, particularly in cotton and rice. Transgenic system of indica rice and wheat have been developed; rice transformed with 3 genes and wheat with one gene to confer stress resistance. A nutritionally important gene AmA1 isolated from Amaranthus has been successfully transferred to Potato. India is now a partner in the International Rice Genome Programme with the responsibility to sequence a part of Chromosome 11. Bt. Cotton trials at several locations have been monitored. The data indicates that Bt. Cotton has an average yield advantage of around 4.0 times over non-Bt. Cotton. No effects of Bt. Protein have been noticed on non-target and beneficial insects. Plant Tissue Culture : Protocol standardization has been completed for a number of species of forest trees, horticulture and plantation crops. Different crosses have been developed in a variety of orchids. Field demonstration trials are being conducted for tissue culture raised coffee, pepper and tea. Complete regeneration systems are now available for 20 different species. Transformation system has been developed forPopulus species. A Polymerase Chain Reaction (PCR) based molecular diagnostic kit has been perfected for the detection of Banana Bunchy Top virus. The Micropropagation Technology Parks at TERI, New Delhi and NCL, Pune have produced 10 million plantlets field planted over an area of 6500 ha at various locations in 17 States. Medicinal and Aromatic Plants : A network of four Gene Banks has been set up for ex - situ conservation of rare, endangered and commercially important medicinal and aromatic plants. About 27,000 stem cuttings of Taxus have been raised and 1500 plants established in natural habitat. Agrotechnology package has been developed for a new variety of Artemisia annua containing 0.5 - 1.3% artemisinin and yielding up to 80 kg of artemisinin / ha. An isoquinoline alkaloid, berberin, which acts as an immunomodulatory agent has been identified and isolated from Berbris aristata (Berberry). A herbal product is being developed for burn cases. In vitro bioscreens have been developed for assaying the anti-proliferative and anti - diabetic medicinal plants. A Mission progamme has been initiated for the development of herbal products with the help of biotechnology tools. Bioprospecting and Molecular Taxonomy : Biome maps for North-Eastern and Western Himalayas have been prepared. Taxus and its associated species have been mapped in Talle Valley of Arunachal Pradesh. A Biomonitoring System has been evolved. The documentation and characterization of ten identified species of the two hot spots has been completed. Molecular characterization of identified economically important species is being carried out. A cold related gene has been identified from a plant species in Spiti region of Himachal Pradesh. 23 salt tolerant gene have been isolated and cloned from the Mangrove region in Western Ghats. Anti-pesticidal fractions have been identified from a tree species for resistance against Cotton Boll worm; 88% growth inhibition recorded. Agriculturally important bioactive compounds have also been identified from five species of the Kumaon and Garhwal Region. National Bioresource Development Board : Under the National Bioresource Development Board inventorisation, characterization and sustainable development of the bioresources of various agro-climatic zones and fragile ecosystems of the country has been undertaken. Animal Biotechnology : Transgenic mice expressing neomycin resistance gene, green fluorescent protein gene and other genes have been produced. A buffalo chromosome probe for sex determination of embryo is being applied in the field. Rabies vaccine for animals using vero cell culture is being validated before commercialisation. Conjugates are being evaluated in female dogs for the efficacy of recombinant dog zona pellucida glycoprotein to regulate fertility. Aquaculture and Marine Biotechnology : A PCR based diagnostic method for white spot syndrome virus of shrimp and vaccines for fish pathogens against infection ofAeromonas sp. and Pseudomonas sp. have been developed. A process has been perfected for the preparation of pathogen free fermented silkworm pupae silage to act as an ingredient in the animal feed. A feed unit with a capacity of 250 kg pellets per hour has been established at Central Institute of Freshwater Aquaculture. Some bioactive compounds from marine organisms are under development. Medical Biotechnology : A therapeutic immunomodulator for Leprosy is now available as ‘Leprovac’ in the market. Genetically modified live cholera vaccine has completed Phase-I clinical trials in humans with no side effects. The anti-fertility vaccine to control fertility in women has been found to produce specific antibodies at a desired level. Steps are being taken for pilot scale production of Rotaviral candidate vaccines for Phase-I clinical trials in the country. For Japanese Encephalitis, efforts are on to produce the candidate vaccine to serve as a replacement for the mouse brain derived JE vaccine. A DNA sequence has been identified for its potential use as a vaccine for Rabies. A recombinant BCG carrying a gene for Microbium tuberculosis has been developed. The recombinant proteins and peptide based candidate vaccines are awaiting clinical testing for malaria. Fourteen diagnostic technologies like IgM, Mac ELISA assay systems for detection of Dengue, Japanese Encephalitis & West Nile; ELISA system to measure alpha-fetoprotein levels in women; IgM based test system for detection of Hepatitis- ‘A’ virus and urine based ELISA systems for detection of four reproductive hormones have been validated. Two diagnostic test systems developed for HIV have been transferred to industry. Goat hepatocytes were found at Deccan Medical College, Hyderabad to be more compatible as compared to porcine hepatocytes for transplantation in patients with acute fulminate hepatic failures in humans. Human Genetics and Genome Analysis : Novel mutations in gene responsible for retinoblastoma at L.V. Prasad Eye Institute, Hyderbad provide help in screening and counseling of patients and their affected families. A multiplex Polymerase Chain Reaction (PCR) is being used to provide diagnosis and counseling in Spinocerebellar ataxias. Sixteen genetic diagnosis cum counseling units have been set up in the country for prenatal diagnosis and counseling for major genetic disorders. Almost fourteen thousand affected families have benefited from this programme. Around 2000 tribals have been screened for detecting various genetic disorders. A National Bioethics Committee has formulated ethical policies for research on human genetics and genomics. Microbial and Industrial : Several microbial strains have been isolated for leaching of metal ores of Copper, Zinc and Gold. Microbial polysaccharide gellan and xanthan have been produced in high concentration using Sphingomonas sp. Production of cyclodextrin has been standardised using microbial strains of Bacillus firmes and Klebsiella pneumoniae. Technology for the bioremediation of Manganese and Zinc mine dumps has been developed through integrated biotechnological approach. Technologies for the production of xylanase, inulinase, lipase and protease have been developed. Patents on cephalosporin process and rifimycin-shuttle vectors have been obtained in India and USA. For xanthan gum, an indigenous production process as an import substitute developed at Birla Institute of Scientific Research, Jaipur has been transferred to Sriram Biotech Ltd., Hyderabad. A programme on microbial biodiversity for accession of important microorganisms has been initiated. Biological Pest Control : Technologies for the mass production of candidate biocontrol agents, baculovirus, parasites, predators, antagonists, fungi and bacteria for economically important crops have been transferred to industry. The important crops include coffee, cotton, oilseeds, pulse, spices, sugarcane, tea and vegetables. A crop area of 65,000 ha has been covered under biocontrol / biopesticide efficacy demonstration. Use of twelve candidate biopesticides has been successfully demonstrated in different agro-climatic zones. Baculovirus based technology has been developed for management of forest insect pest (teak defoliator). Integrated Pest Management module has been developed for green (organic) cotton under irrigated conditions. Fermentation based technologies of three fungal biocontrol agents have been transferred to industry. New potential biopesticide technologies are being revalidated for commercialization. Biofertilisers : Mass production technologies for Azotobacter, Azospirillum, Blue Green Algae (BGA), Mycorrhiza and Rhizobium have been developed and transferred to industry for production and marketing. It has been found that Rhizobium biofertiliser can save upto 25-30 kg chemical nitrogen in pulses and leguminous oil seeds. Azotobacter, Azospirillum and Mycorrhiza can make phosphate available in soluble form with other micronutrients to most of the commercial crops. Around 10,000 demonstration programmes benefiting about 35000 farmers have been conducted. Biodiversity Conservation and Environment : Technology package for eco-restoration of mine spoil dumps and phytoremediation of dye industry effluent has been standardized. An efficient crude oil and oil sludge degrading bacterial consortium 'Oil Zapper' and biobeneficiation and desulphurisation technologies have been transferred to the industry. Seribiotechnology : A programme on silkworm genome has been initiated at the Centre for DNA Fingerprinting and Diagnostics, Hyderabad. A technique has been standardised for effective degumming of silk using fungal protease enzyme for better lusture and softness of dupion silk. Field trials of immunodiagnostic kits have been conducted at farmers' level for early detection of infectious flatcherie disease. More than 100 microsatellite DNA markers have been identified to be used for mapping genes of silkworm. Eight molecular RFLP markers linked to the cocoon shell character of the silkworm have been identified. They are likely to be used in directional breeding programme. Expression of marker genes has been demonstrated in silkworm. Silkworm is likely to be used as a bioreactor in future. Gynogenic haploids have been produced in mulberry and are being field evaluated. Food Biotechnology : Low cost nutritious food supplements for school going children have been developed. Large-scale production of Betalains (food colorant) and Oyster mushroom optimized. Process for preparation of clarified fruit juices with aroma retention and food flavours developed. Diagnostic kits for rapid detection of food borne pathogens have been developed. Repositories and Biotech Facilities : A large number of infrastructure facilities and repositories have been set up in different universities and research institutes to facilitate advance research. The facility at IMTECH, Chandigarh for the microbial type culture collection has now been upgraded to an International Depository Authority (IDA). The repository on Drosophila has been strengthened to develop educational kits for demonstrating genetic experiments in schools and colleges. Studies at the repository on filaria have resulted in patenting of a unique filarial antigen. Genetic engineering and strain manipulation has demonstrated over production of daunorubicin and anti-cancer antibiotic at pilot scale. A national containment facility has been set up at NBPGR, New Delhi to look into various quarantine issues relating to transgenic planting materials. A National Facility for Virus Diagnosis and Quality Control in Tissue Culture raised Planting Materials has been set up at Indian Agricultural Research Institute, New Delhi with six satellite centres. This facility will certify all domestic and export consignments. Bioinformatics : India is one of the first countries in the world to initiate a Bioinformatics programme. A National Bioinformatics Network has been evolved for sustainable utilization of the biological data resources. The 56 centres, six interactive computer graphics facilities and four post graduate diploma courses in Bioinformatics are being supported. Internationally recognized databases such as EMBnet, PDB, GDB, EBI and Plant Genome database have been established in the form of Mirror sites under the National Jai Vigyan S&T mission. These databases are being utilized for genomic and proteomic R&D activities through high speed and high bandwidth network services. Human Resource Development : With sixty two biotechnology teaching programmes in various Indian universities and institutions about 1600 students have been trained in two years. These include a special programme in Neurosciences. National and Overseas Associateships awarded annually to about 45 scientists has resulted in very good expertise. National Bioscience Awards for Career Development and National Women Bioscience Awards are conferred annually to recognize the outstanding contributions of biotechnologists in various fields. Number of biotechnology scholarships given to school leaving students to encourage them to pursue a career in biology related areas has been raised from 10 to 25 per year. IPR and Biosafety : A Biotechnology Patent Facilitating Cell has filed 75 patents. Roving seminars are conducted at regular intervals to create awareness on Intellectual Property Rights and related issues. Biotechnology for Societal Development : Training, large scale demonstration and cooperative marketing activities in the areas of food processing, leafy vegetable and mushroom cultivation, vermicomposting, organic farming, orchids, ornamental plants, conservation of rare and endangered herbal medicinal plant species have benefited this target group significantly. Sericulture has been introduced in non-traditional areas for economic upliftment of women. A Women’s Biotechnology Park with many industrial modules at Chennai is a success. Biotechnological interventions for disaster management have given rich dividends. Biovillage : A Biovillage at Mocha village, Porbandar for extension of technologies which are pro-poor, pro-women and pro-nature to the grass root level has been a model for rural development. Farmers of target village have been trained in production and use of bio-fertilizers, bio-pesticides, modern farming practices and cultivation of marine algae seaweeds. Cultivation of medicinal and economically important plant species has been undertaken in the wasteland of the village. A desalination unit for pure water has also been set up. Autonomous Institutions : The Department has set up following autonomous institutions for achieving excellence in specialized areas which have been further strengthened : National Centre for Plant Genome Research, New Delhi National Brain Research Centre, Gurgaon. These institutes are working in the frontier areas of New Biology and Biotechnology, have a high degree of excellent publications to their credit and many research leads have been converted into products and processes and transferred to industry. Recently the institute for Bioresources and Sustainable Development has been established at Imphal under the auspices of the National Bioresource Development Board. Biotechnology in Different States : The dialogue is on with various States for developing initiatives in biotechnology, specially to deal with location specific problems. Jai Vigyan National Science & Technology Missions : Four mission projects implemented by DBT have made significant progress. These are: Generation of new vaccines; herbal product development, coffee improvement and establishment of mirror sites. International Collaboration : Under the Integrated Long Term Programme of Indo-Russian collaboration in biotechnology an Indo-Russian Centre for Biotechnology is being established. A new phase of Indo-Swiss collaboration in biotechnology is under implementation during 1999-2004 with focus on biotechnology applications in agriculture and environment with special reference to improvement of wheat and pulses. Vaccine and contraception, brain research and genomics are the areas for joint programmes with USA. More than a dozen new collaborations both with developing and developed countries have been initiated. Thus, biotechnology research and application are moving in a new direction for creation of a strong knowledge base and for conversion of this knowledge into economic and societal benefit.