Tuesday, May 27, 2008

Is Synthetic Biology Really New, or Old Wine in a New Bottle, or harbinger of a Brave New World?

Sohan P. Modak
May 27, 2008
Henry Harris, in 60s, developed the technique of cell fusion to form heterokaryons by fusing nucleated chicken erythrocyte with Hela cell by assisting the cell fusion by UV-inactivated Sendai virus. Heterokaryons contain genetically different nuclei in a single cell. In contrast, homokaryons are produced by fusing two cells of the same genotype giving rise to bi- or multi-nucleate cells. This principle was exploited by Rao and Johnson [http://www-rcf.usc.edu/~forsburg/ccslides/sld001.htm] to fuse cells from different phases of cell cycle to examine the effect of intracellular environment on chromosomes of nuclei in different phases of cell cycle (Figure 1).

One of the most important spin-off from cell fusion experiments is the onset of Somatic Cell Genetics, i.e. genetic studies of cells other than germ cells. Somatic cells are diploid and differentiated. Therefore Somatic cells from different tissues of the same individual should be genetically identical but may acquire tissue-specific differences during differentiation. Most differentiated cells do not grow readily in culture as most maintain tissue-specific or differentiated traits but have lost their ability to divide. Such cell populations may contain a subset that retains division potential but a exhibit finite lifespan, unlike those derived from tumors or transformed in vitro with viruses, e.g. Adenovirus, Epstein-Barr virus, etc. or treatment with carcinogens and become immortal.
Early cell fusion experiments revealed that in hybrid cells, say human and rodent cells, most human chromosomes were selectively eliminated while retaining the rodent chromosomes and establishing a novel karyotype. In case of human-chicken hybrids, most chicken chromosomes were eliminated which allowed pinpoint the chromosomal location of expressed chicken phenotype like isocitrate lyase. Thus cell hybridization technique permits mapping genes to specific chromosomes or chromosomal sets, a forerunner of genome mapping. Another example includes mapping thymidine kinase on human chromosome 17. A huge amount of work was contributed by teams at Karalinska Institute, Sweden.
The next step involved producing chromosome-specific hybrids where in single chromosomes are transferred to cells. By prolonged treatment of cells with colcemid or colchicine, again a technique pioneered by Karolinska group, nuclear membrane reconstitutes around metaphase chromosomes in the absence of mitosis and gives rise to mini nuclei that can then be separated and fused with cells from another species giving rise to hybrids bearing full karyotype of the recipient cell and one chromosome from the donor specie, again allowing specific gene mapping studies.
In 50s and 60s, nuclear transplantation experiments were pioneered by Briggs and King in USA and Gurdon, Blackler, and Fischberg in UK. These demonstrated that intra-generic nucleo-cytoplasmic combinations perform better than those across genera. These experiments opened new frontiers in cell tinkering were when it was shown that and led to preparing enucleated somatic cell cytoplasm which is then fused with a foreign nucleus thereby creating a Cybrid wherein genomic DNA and cytoplasmic mitochondrial DNA are of different origin. Cybrids were developed by treating monolayer cultures with cytochalasin, which depolymerizes the cytoskeleton and the loosened nucleus can then be removed by centrifugation leaving behind cytoplasmic mass. Such cytoplasmic bodies well delimited by the plasma membrane and containing all intracytoplasmic organelles are then fused with isolated nuclei from a different cell type either in the presence of polyethylene glycol, or Sendai virus, or simply microinjected into the cytoplasm. Hence one could form cybrids between HeLa cytoplasm and mouse or chicken or rat nucleus.
This principle is now being applied to exchanging zygotic DNA with that from somatic cell of another species to generate a novel set of hybrid stem cell lines that would have direct applications in replacing diseased/damaged cells in tissues of patients suffering from degenerative diseases or in accident cases.
The first highly successful of protoplast fusion was demonstrated by Carlson’s team in 1972between species Nicotiana lansdorfia and Nicotiana glauca that led to a spurt of activity in developing methodologies to produce viable plant heterokaryons as well as and homokaryons. Bacterial protoplast fusion was first reported by Rollin Hotchkiss’s group in 1976 (Schaeffer et al., PNAS, 73, 2151-2155). The principle claim of these experiments was that, similar to plants, whole genomes could be transferred among bacteria. Thus, intra-stain and intragenic genome transfers with resulting genome hybrids do give stable progeny With this background one should examine the recent advances in genome transfers. In June 2007, Carole Lartigue from Venter Institute transplanted entire chromosome of Mycoplasma capricolum (JVC1.10) into Mycoplasma mycoides (LC) to change the bacterial species phenotype to Large Colony (LC)., and showed transformation one type of bacteria into another type dictated by the transplanted chromosome (C. Lartigue et al., Science, Genome Transplantation in Bacteria: Changing one species to another. Science, 317, 632-638, 2007). In parallel, scientists at Craig Venter Institute set out to construct the entire 582,970 base pair genome (3.6 x 108 daltons) located on a single chromosome of Mycoplasma genitalium (JCVI-1.0), a sexually transmitted infectious agent in humans, and Gibson et al. reported successful completion (Gibson et al., Complete synthesis, assembly and cloning of a Mycoplasma genitialium genome., Science, 319, 1215-1220). Until now it has been possible to synthesize 30,000 base pair-long DNA in vitro as longer fragments are highly susceptible to errors and mechanical damage. According to the JCVI team the copy is perfect, but they voluntarily have modified a gene necessary for the bacteria to infect people. The entire chromosome-length double stranded molecule of synthetic DNA containing specific identification markers that do not affect the information content of the genome, was then successfully transferred to enucleated Mycoplasma capricolum. Clearly CVI team has taken a quantum jump in synthesizing a fully readable genetic script and transferred it into a compatible environment. Here the crucial issue is that the donor genome should be able to take control of the living cell cytoplasm and convert it into a new life form. Basically, what Craig Venter’s team has achieved is to translate the earlier work on intraspecific chromosomal and cellular hybrids to a stage where entirely new species can be created by synthesizing and transferring a artificially written DNA script in an readable form. Craig Venter has now declared that his next step is to synthesize organisms that use atmospheric CO2 to produce methane and reduce dependency on fossil fuels.
One the prime issue facing the Molecular Geneticists concerns the fact that ghe genetic grammar is still not fully understood. For example, while we know the basic triplet code and some of the punctuation marks such as the beginning and end of a genetic coding sentence as well as some of the alphabet blocks/words acting as signaling elements for retrieval of the genetic information and converting it into a polypeptide, there is a huge lacuna in conceiving, let alone understanding the a potentially large number of signaling elements or punctuation marks in form of words longer than triplets which are located in the non-coding regions of the genome. Therefore, transferring a readable genome synthesized in vitro into a closely related (intra-genic) organism is one thing, but creating one that would be compatible to completely different intracellular environment and control take over the biochemical and biological processes leading to survival and replication is a dream for the future. In the interim, however, the time has come to produce designer organisms that are otherwise very closely related to the existing ones but more useful. For example, would it be possible to synthesize a non-virulent strain of Mycobacterium tuberculosis that competes out the virulent strain? Such are the possibilities of Synthetic Biology. Needless, to caution that there are always individuals , organizations or even Governments that could use this technology for Biological and Economic Warfare! Whatever the ethical and socio-considerations, successful generation of new species would require far detailed understanding of the sthat ignals that govern allele-specific, gene battery-specific and temporal and positional regulatory signals and signal networks that qualitatively and quantitatively control specific gene expression. Well, we are still far away from that stage. For example the present methodology of gene annotation is at best tedious, clumsy as it requires knowledge of the amino acid sequence of the final product of gene expression before it can be retraced via bottoms-up approach to the genome. It is a trap that has led searchers to wild goose chase for a bona fide coding sequence from among many partials, homologues, Paralogues and requiring expensive and time consuming validation by wet lab analyses.
Can we not think of a vastly different Top-down approach to develop a gene annotation methodology?

Saturday, May 17, 2008

The wet option

The design of life is the quest of synthetic biology- Don't all approaches to design carbon based life rest on the central dogma of molecular biology? SPM, I guess you would you like to contest that?

Here is one approach to designing life: Craig Venter at his laboratory promoting wet (organic) synthetic life.

Pranav, can we rake up a discussion saying GenePython offers steps to the another approach: your own laboratory of artificial life, on your computer?

Is it a cool way to create building blocks of genes and cells and extend the functionality of genes, RNA and peptides as you wish and to keep pace with the stalwarts of web-biology? Is it a safe way to play with lifes building blocks, an easy way to learm about the central dogma and maybe even do thought experiments challenging the dogma. Is it also a great way to design, recognize and explore feedback based regulation?

So before we get going with explaining GenePython, can we say it's a framework to start building life designs and do what you want out of it! Right Pranav?

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About Gene Python...

After thirty years of the central dogma we are now in an age where we can create an in silico cell and simulate the whole of the central dogma!

Ever wanted to do gene engineering and thought it took weeks? Ever wanted to figure out gene expression and found the painful wet labs to drive you nuts? Ever wondered what peptides could be generated from a molecule of mRNA? Or just plain wanted to have fun putting together genomes and having them generate peptides and proteins? Wanted to create an automatic gene annotater?

What fun it would be if we could assign a gene to a computer variable and simply let the computer behave as if it were the central dogma and generate the RNA molecules and convert those with reading frames into peptides! If only we could explore the wonders of nature in minutes and map the proteins or peptides to known ones from published databases! What if we could use such designs to genetically re-engineer real genes in organisms?

GenePython initiates a journey to make this exploration fun. It is the result of intense dialogue on the central dogma and an effort to create genetic objects in python over a few weeks in November and December of 2007.

This blog and this site are our effort to get you to join the fun and come and build a virtual cell together with us. Over the next blogs we will expose the code of genetic objects to help you understand and begin use of these objects as building blocks of the cell.

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