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Challenging on transcriptome
Posted on July 2nd, 2010 No commentsmRNA translation is one of crucial process in cellular metabolism. Several approaches have been developed by cells to perfectly regulate the translation. microRNAs bind mRNA and determine its half life, while numerous proteins directly bind the mRNA and favor or avoid the interaction with ribosomes.
Studying the interaction between proteins and mRNAs is important to well understand whether alteration in these binding sites have any contribution to genetic disease that are caused by a loss of an RNA- binding protein such as fragile X mental retardation or familial amyotrophic lateral sclerosis. Scientists form the Rockefeller University described a method to isolate complexes protein- RNA. They used the UV radiation that are known to form covalent bonds between protein and mRNA. So, live cells are exposed to UV radiation, and an immunoprecipitation against the protein binder allows to identify the sequence involved in the binding. This technique called CLIP (PDF), cross linking and immunoprecipitation, can be applied also to high throughput experiments. To do this some improvement are necessary in order to separate the signal from background. The use of photo- activable ribonucleotides introduces into the RNA some non-toxic and efficient linkers. Indeed, a specific base change during reverse transcription, scoring for thymidine to cytidine in the sequenced cDNA allows to precisely map the interaction site. This is an important step to unravelling the gene regulation. -
The whole genome sequencing to identify Mendelian disorders
Posted on June 9th, 2010 No commentsThe current way to determine the cause of disease is finding mutations via DNA sequencing. In order to reduce costs only coding regions are sequenced and analyzed. Unfortunately, several Mendelian traits that can be the basis for specific diseases are not present in coding regions.
Therefore, the sequencing of the whole genome might contribute better understand the causal variant of diseases. Scientists from the Institute for Systems Biology in Seattle proposed this approach to study the Mendelian hesitance of two recessive disorders. They analyzed the whole genomes of healthy parents and sick children. They delineated an accurate recombination map showing exactly which pieces of parental chromosomes had been assembled in offspring genetic material. Then, they corrected 70% of sequencing errors and especially they reduced the search space for the disease- causing variants. This study is important because it demonstrated that is possible to identify the genes involved in etiology of certain disease by sequencing the DNA of the family in which this disease appears. Based on this observation, scientists plan to analyze the genome of family with Huntington’s disease. This approach requires the absolute precision of sequencing data. -
The synthetic genome
Posted on May 28th, 2010 No commentsThis is the news of the day: scientists fro the Craig Venter Institute generated the first genome of a viable cells. The research has been published on Science Express and in few hours has been diffused and commented around the world. What is the story of this clamorous paper? Scientists used one yeast strain as a model. Firstly, they in silico designed the genome and placed some sequences of control in order to surely distinguish synthetic genome from the natural one. Previous experiences in sequencing helped researchers to well design the synthetic genome. Indeed, we have to remember that the Craig Venter Institute was one of the first institutions to complete genome sequencing of several organisms.
They spitted the synthetic genome into small portions and sequentially assembled them. They started from 1kbp units that were amplified in E. Coli, purified and transformed in yeast. Ten 1kbp formed the first 10kb intermediates. In order to control the intermediates quality, multiplex PCR was carried out, by using specific primers that bound the connecting sequences. They repeated the same process also to produce 100kb intermediates, but they directly amplified the DNA in yeast because E. Coli wasn’t able to do it. The final assembling required additional vector sequences and was performed in yeast spheroplasts. The main issue of this step was the removal of natural genome: synthetic DNA was trapped out from agarose plugs and digested. The synthetic genome was then transplanted into host yeast. In the discussion authors outlined how was important the quality control of each step. Indeed, none mutations had to be introduced into the synthetic genome, especially in gene crucial for cell viability. Indeed, they explained that lost several weeks because of a mutation in DNA gene. The novelty of this paper is the capability to produce a synthetic genome compatible with cell life and propagation. Indeed, the technology to produce short or longer sequences, i.e. plasmid, is quite common and already commercially available. In this case, yeast still is able to duplicate and the genome is pretty more complex than a plasmid. Informatics skills to predict the correctness of the final genome are essential to successfully accomplish this project. Finally, some ethical concerns about the opportunity to manipulate genes at this point and generate life arise from similar studies. Authors self invite the public opinion and other scientists to continue the debate about ethics. Indeed, we must keep in mind that the final goal of science advances is the human life improvement, in terms of quality, health, environment and similar researches may have important advantages for overall world and us. -
Ten years after the Human Genome Project
Posted on April 6th, 2010 No commentsThe Human Genome Project started ten years ago, with the challenging promise to sequence the whole genome and definitively understand all genetic secrets.
Two astonishing –but also scientifically interesting- surprises were presented to scientists of all countries: firstly only few genes are present in the genome, and this number -20000- is not so different from those of other species; secondarily, the major portion of DNA has regulatory functions rather than encoding significance. Thus, human genome sequencing has generated a lot of further questions about the mechanism of expression tuning. In 1960s Jacob and Monod demonstrated the presence of gene regulator in prokaryotic organisms, such as E. Coli; only few years ago we obtained the confirmation of this presence also in the human genome and numerous gaps have to be filled to reach a comprehensive understanding of molecular mechanism in cells. Epigenetic studies, microRNAs identification and gene expression analysis will help to gain a complete overview of human genome regulation, in addition to gene sequencing. Moreover, the Proteome Project will continue to clarify how proteins are involved in cellular life. Fortunately, a lot of open questions still be unsolved, and a lot of work has to be done by scientists worldwide. -
The relationship between thalassemia and atherosclerosis
Posted on March 9th, 2010 No commentsSardinia is an Italian island where thalassemia incidence is really high. Giving the complications of atherosclerotic disease and the necessity of frequent blood transfusions for thalassemic patients, scientists from the University of Cagliari (Italy) were interested in the relationship between these two diseases.
They provided a genetic analysis of patients affected by thalassemia major or intermedia in comparison with age matched healthy controls, by checking genes involved in iron detoxification and in cholesterol metabolism. Indeed, cholesterol levels are a crucial factor to determine the atherosclerotic lesions as well as iron overload, especially in heart, is important to cause cardiovascular complications. They identified an increase of TNFa and ACAT mRNA levels, involved in iron metabolism and cholesterol metabolism, respectively and a reduction of Hepcidin and ILa. Serum iron levels were higher in patients than in control, while HDLs were lower. Since gene expression was altered in factor that had a key role in cholesterol metabolism rather than in iron homeostasis, scientists suggested that possible cardiovascular complications in patients affected by thalassemia intermedia were due, at least in part, to the occurrence of premature atherosclerotic lesions. By contrast, the role of iron overload was further confirmed in thalassemia major patients. This preliminary study allows clarifying how relationship between important diseases, such as thalassemia and atherosclerosis, may contribute to complicate the clinical profile. -
From a protein to pharmaceutical target
Posted on December 30th, 2009 No commentsCurrent pharmacological approaches to identify target have strong molecular basis. Giving the important results of genome and proteome projects in understanding cell biology and biochemistry, also pharmaceutical research changes its approach and obtains great advantages from this new knowledge. Indeed, once identifying mutations in genes involved in particular diseases in the genome project, the role of proteins have been defined by the proteome project. Numerous proteins responsible of cancer, neurodegenerative disease, immune disease and so on have been studied in this way, obtaining important results in disease understanding.
The first step to treat one disease is knowing the pathological mechanism on its basis. Altered proteins could have a high activity, in this case we talk about gain of function mutations, or lower activity than normal, and so we talk about loos of function. In both cases, genetic alterations cause an impaired protein activity with deleterious consequences on cellular homeostasis. One protein acquires pharmacological significance when has a crucial role in disease arising. The common way to demonstrate the importance of one protein in pathogenesis is silencing or restoring it, in the case of gain or loss of function respectively. SiRNA approach is often used to silence proteins and analyse the effect of this lack in cellular system. The big advantage of siRNA is the selectivity: indeed, if siRNA is well designed, it’s possible to obtain complete depletion of the protein of interest without interfering with other cellular functions. So, this approach is safer than the use of chemical inhibitor because of the lack of collateral events. Furthermore, siRNA is applicable to every kind of protein even if their function has not been clarified yet. In the case of loss of function, it’s possible to restore protein function by transfecting cells with wild type version of the protein. Unfortunately, this system is not so theoretically universal like siRNA, because in the case of dominant negative mutation the normal protein function cannot be restored. Numerous animal models are available to confirm results obtained in cellular assays: knock out and knock in animals have been developed to simulate loss or gain of function, respectively. In summary, in cellular system we can define the role of a protein in the disease of interest and we can understand how it’s possible to modulate its functionality in order to restore normal homeostasis. Then, in animal model we can verify the consequences of proposed treatment in a whole organism. This is the way to transform a simple protein into pharmaceutical target. Basic studies on protein function are usually performed in public institution or universities, in some cases with the contribution of pharmaceutical sponsor that would have major benefits from research results. Otherwise, the presence of spin off or start up companies inside the university allows preserving the economic value of research for the institute. In this manner, research independency is guaranteed and also diseases which pharmaceutical companies are not strictly interested in, could be studied. -
Novel mouse model for BRCA1 breast cancer
Posted on December 9th, 2009 No commentsBRCA1 is one important marker for breast cancer; in particular BRCA1 is mutated in the invasive ductal cancer and in general is associated with lack of oestrogen receptor, progesterone receptor and Her/ ERB2, making useless current therapies for breast cancer. Giving the lethality of double mutant Brca1, none animal models were available up to date to study the role of this gene in tumorigenesis.
Everyone knows the importance to study cancer from its molecular basis. Scientists from the Netherlands Cancer Institute of Amsterdam proposed a conditional mutant in which tissue specific inactivation of Brca1 was performed by Cre Recombinase. This system allows to dispose on one new model that mimics several characteristics of human mammary tumour. Furthermore, numerous new inhibitors or useful molecules could be tested on this model in order to find out some drugs to care BRCA1 cancers. The work, published on the British Journal of Cancer, open new perspectives in breast cancer research. Indeed, the next generation of mouse models will regard resistance mechanisms and double mutant animals will be likely generated in order to highlight the correlation between lack of BRCA1 and the role of other proteins often mutated in breast cancer, such as TP53. -
Gene therapy to restore sight
Posted on November 2nd, 2009 No commentsLeber’s congenital amaurosis is a pathology that affects 3000 people in USA and causes loss of sight from birth until reaching complete blindness at about 40 years. This disease interests a gene, RPE65 that helps in rhodopsin synthesis. If this gene is mutated, as occurs in the disease, photoreceptor cells into the retina will die.
During an important study carried out at the University of Pennsylvania, gene therapy was applied to blind dog to restore the activity of RPE65 gene and restore sight, as a consequence. Then, a small- scale study in human started in 2007, to ascertain the safety and efficacy of using virus to carry the RPE65 gene. First results showed that four of six young adults with Leber’s congenital amaurosis, had an improvement in their sight. Nevertheless, researchers knew that children with an intact retina –not already compromised by the disease- could have major benefits from this therapy. Thus, they performed the same study including four children for 8 to 11 years old and obtained great results in terms of increased light sensitivity. This work has been published in Lancet Journal in October, 2009. As all therapies that can improve the life quality of children, this work offers an hope for families that have so serious problems. -
New technologies to identify gene function
Posted on October 30th, 2009 No commentsAfter advances in DNA sequencing technology, the major task is to determine the functional role of proteins coded by these sequenced genes. Given the broad range of different functions carried out by proteins, it’s obvious that a multiplicity of techniques will be necessary, while DNA sequencing is achieved by few, easy and simply technique. A series of strategies based on generalization and systemization of genetics are emerging now as important tool to fill the gap between sequence and activity. One of these approaches is the analysis of the effect of perturbations of gene expression, by deletion, mutation or over-expression: after one of these modifications, we expect to observe a phenotypic change.
The challenge is to quantitatively measure phenotypes with enough accuracy and depth to define gene function. Two complementary approaches for determining complex phenotypes are currently used: in the first one many different parameters are simultaneously analysed, this is an high content screen; otherwise a single or limited number of aspects are observed, but the effect of perturbing each gene is followed in combination with a second perturbation, either another mutation or a chemical treatment. This genetic interaction profiling offers a high-resolution view of the function of each gene. Saccaromyces Cerevisiae is a model really useful for this kind of studies: a complete series of deletion strains of nonessential genes has been produced and has allowed to better understand the role of proteins important for yeast biochemistry and biology. Important results have been achieved also by using conditioning mutants that selectively grow in rich media: also in this case precious information has been retrieved. Rather than loss of function studies, methods for systematic gene over-expression have been optimized. Novel approaches in this field are interested in construction of untagged proteins in order to exclude that the presence of tag could interfere with the normal function of protein self.
The main goal of this systematic studies is to maximize the information flow, while minimally compromising the accuracy of phenotype detection. The introduction of large biomolecules into cells, such as DNA, RNA allows to directly analyse the role of one gene in the cellular life, and different kind of cells (mammals, primary cells, stem cells) can be used in this approach. Biomolecule is printed in an array onto glass slides, as done in conventional microarray. A monolayer of cells is deposited on top of the arrayed molecules and cells are transfected by taking up the material from glass. By using 96-well format plate, it’s possible to analyse the effects of a large number of biomolecules in a quantitative way. A plausible example of this method application is the effect of iRNAs on cellular proliferation: iRNA can be printed on bottom plate, cells are transfected (please note that is important to define the efficiency of transfection) and proliferation rate can be measured with normal treatment with MTT. In this way, genes important in proliferation could be identified.
Future efforts will be done to exploit a vast array of data that will emerge from large-scale genomic and proteomic projects to gain a deeper knowledge of the function of biological system. -
Manipulation of bacterial genome in yeast
Posted on October 28th, 2009 No commentsEven if manipulation of bacterial genome is often difficult and challenging, engineering allows to better understand bacterial biology and genetics. Researchers from C. Venter Institute improve a protocol to clone bacterial genome in yeast, manipulate it and boot it up in bacteria self. To do this they chose an “easy” model, Mycoplasma, because this organism doesn’t have bacterial wall, its genome is small and A-T rich, so is more properly replicated in yeast than ones rich in G-C. Furthermore Mycoplasma has non-standard genetic code that can not be translated in yeast, preventing the synthesis of bacterial proteins toxic for yeast.
What did scientists perform to achieve this important result? They cloned Mycoplasma genome into yeast artificial chromosomes (YACs), genetically manipulated it and then transplanted it into the final organism receiver. Two concerns could prevent this goal: one was the possibility that restriction endonucleases recognised foreign sequences and degraded them and the second one was that yeast modified bacterial genome. Fortunately this last event didn’t occur, while to limit endonucleasic activity, scientists hypermethylated donor genome and eliminated endonucleases from receiver organism. This protocol could be improved in order to become a conventional technique for bacterial manipulation in order to have another tool to solve human needs in medicine and environmental preservation.
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