The Life Sciences Today

The biological sciences have experienced enormous growth over the last century,

fueled by a stream of discoveries — such as the principles of genetics, the structure of

DNA, and the discovery of gene splicing technologies. These have opened new fields of

inquiry and provided the basis for myriad applications in industry, agriculture, and

medicine. Among the technological breakthroughs in the life sciences, genetic

engineering plays a particularly significant role.

Genetic engineering is a technique that permits the artificial modification and

transfer of the genetic material from one organism to another and from one species to

another. This technology is used throughout the world to alter the protein produced by a

gene and to design organisms with desirable traits for applications ranging from basic

research and development activities to pharmaceutical and industrial uses. During the

last 30 years, these recombinant techniques have spawned a vibrant biotechnology

industry focused largely on the development of new pharmaceuticals to fight disease.6

By 2000 the annual investment in the biotechnology industry peaked at nearly $29

billion, while employment in the biotechnology industry reached 191,000 by 2001.7

In response to the opportunities presented by these developments the resources

devoted to the life sciences have increased dramatically, making further discoveries

possible. The government has funded biological research generously through the

National Institutes of Health and National Science Foundation budgets, with few strings

attached; private foundations and the pharmaceutical industry have also made major

contributions. The number of PhDs awarded each year in the biological and agricultural

sciences has increased steadily; 6,526 were awarded in 2001.8

This ever-expanding research activity has resulted in numerous new

biopharmaceutical products that are transforming medicine. Examples include human

recombinant insulin for the treatment of diabetes, a vaccine against hepatitis B, and

medicines for diabetes, cancer therapy, arthritis, multiple sclerosis, cystic fibrosis, heart

attacks, hemophilia, and sepsis. As knowledge of the human genome increases, it may

even become possible to tailor pharmaceutical products not only to specific diseases but

also to specific individuals. Throughout this process, the time between new discoveries

and their applications has grown ever shorter. One example is the very short time it took

the scientific community to identify the coronavirus as the causal agent of the newly

emerging human disease, severe acute respiratory syndrome (SARS).

Biotechnology research is now a truly global enterprise. While industrialized

countries such as the United States, the United Kingdom, Germany, Israel, and Japan

may be the first to develop advanced research and technologies, other countries have a skill base that will enable broad domestic utilization of biological technologies.9 For

example:

China has an aggressive program in plant biotechnology, and as of 2002

plans to increase funding by 400 percent by 2005. This energetic

investment also exists in the Chinese private sector, and the national

scientific establishment is attempting to lure foreign-trained scientists to

return with lucrative financial packages. India is in the process of tripling

funding to its national biotech center, and is promoting the development

and use of genetically modified crops throughout Asia. Singapore has for

many years made a practice of recruiting foreign scientists. Taiwan is

investing large amounts in biotechnology and is seeking citizens to return

home to build up biotechnology in academia and industry. A Brazilian

coalition recently demonstrated sophisticated domestic use of biological

technologies by successfully sequencing the plant pathogen X. fastidiosa in

2000.10

In addition to the dispersed research enterprise, publications and personnel are

also widely spread. Well over 10,000 journals in the life sciences are published

worldwide. Biological Abstracts, an international database on biology, clinical and

experimental medicine, biochemistry, and biotechnology, provides coverage of over

6,000 active international journals and 14,000 archival titles from over 100 countries;

Medline, the online service of the National Institutes of Medicine, provides abstract

information for more than 4,600 biomedical journals published in the United States and

70 other countries; and PubMed currently provides full-text web access to 4,058 journals.

According to Medline, the total number of scientific articles published in the peerreviewed

biomedical literature has increased from 449,109 in 1998 to 491,620 in 2001.

Given the global nature of the biotechnology research and development enterprise, it is

unrealistic to think that biological technologies and the knowledge base upon which they

rest can somehow be isolated within the borders of a few countries.

The rapid advance of scientific knowledge and applications owes much to a

research culture in which knowledge and biological materials are shared among

scientists and people move freely between universities, government agencies, and

private industry. Large numbers of foreign graduate students and postdoctoral

associates have been an essential ingredient in the success of the biological research

enterprise. The scientific workforce is increasingly international; at the National Institutes

of Health, for example, approximately 50 percent of the technical staff are non-U.S.

citizens. Research results have been widely disseminated, so that even high school

students now routinely perform experiments involving recombinant DNA techniques. In

short, a dynamic national and international research enterprise has evolved, with an

extraordinary record of achievement at multiple centers of excellence. These are values

that should be preserved in any sensible policy for minimizing the risks associated with

the misapplication of the fruits of the biotechnology enterprise.

MOLECULAR BIOLOGY

The great achievements of molecular biology and genetics over the last 50 years

have produced advances in agriculture and industrial processes and have revolutionized

the practice of medicine. The very technologies that fueled these benefits to society,

however, pose a potential risk as well — the possibility that these technologies could

also be used to create the next generation of biological weapons. Biotechnology

represents a “dual use” dilemma in which the same technologies can be used

legitimately for human betterment and misused for bioterrorism.

Events over the 1990s focused growing attention on this balance of risks and

benefits, part of a larger concern about the proliferation of weapons of mass destruction

(WMD) — chemical, nuclear, or biological. In early 1992, President Yeltsin

acknowledged that, despite being an original signatory and State party to the Biological

and Toxin Weapons Convention (BWC), the Soviet Union had maintained a major

clandestine biological weapons program into the early 1990s.1 Yeltsin ordered the

program shut down, but concerns about other possible secret programs remained.

Policymakers in the United States became increasingly concerned that so-called ”rogue

states” would turn to WMD to counter the overwhelming U.S. conventional military

superiority. Secretary of Defense Les Aspin launched the “Defense Counterproliferation

Initiative” in December 1993 to develop additional means to address these threats.

Official statements continue to cite at least a dozen countries believed to have or to be

pursuing a biological weapons capability.2 U.S. and British concerns about Iraq’s

reported biological and other WMD programs were a primary reason for launching

preemptive military action to find and destroy these weapons capabilities.3 The terrorist

attacks of September 11, 2001 and the subsequent anthrax letters accelerated already

existing concerns that terrorists would seek WMD capabilities as well. President Bush, in

a speech at West Point in 2002, said: “The gravest danger to freedom lies at the perilous

crossroads of radicalism and technology. When the spread of chemical and biological

and nuclear weapons, along with ballistic missile technology — when that occurs, even

weak states and small groups could attain a catastrophic power to strike great nations.”4

States, groups, and individuals are pursuing a biological weapons capability — and the

means for them to do so are widely available.

Biological weapons have long been stigmatized as “indiscriminant agents of

unnecessary suffering, [whose] use … contradict(s) the universal principles of war.”5 As

discussed below, since November 1969 the U.S. programs linked to biological weapons

have been restricted to research and development on defensive measures only. Thus

few biologists in the United States today have knowledge of our country’s past offensive

weapons programs or of the concerns of the national security branches of government.

In this respect the life sciences community is in a different situation from that of the

physics community, which in large part has been continuously involved in governmentsponsored

weapons research programs since at least World War II. The scientific

community and the government jointly face a double challenge: (1) to establish a

working relationship with the national security branches of government, and (2) to help

craft a system that will minimize the risk of wrongful use of biological agents or

technology without damaging the scientific infrastructure that has made biological

research so vital to the health of the nation.

microRNAs aid reprogramming

microRNAs specific to mouse embryonic stem cells can substitute for the reprogramming factor cMyc in the generation of induced pluripotent stem cells. The development of reprogramming methods that do not rely on transgenes may facilitate clinical translation of this technology.

Synthetic networks by design

Strategy for rationally assembling gene networks with predictable behaviors. Using mathematical models, they predict the responses of complex synthetic gene networks built from quantitatively characterized promoter libraries, and harness these networks to regulate an industrially relevant yeast phenotype.

New Initiative to Study Societal Issues Associated with Synthetic Biology -- A Rapidly Developing Field where Novel Organisms are Constructed from The

NEW YORK, Dec. 18  -- The Alfred P. Sloan Foundation announces a new initiative to study societal issues associated with synthetic biology -- a rapidly developing scientific field where researchers are constructing novel organisms from the building blocks of DNA. This new effort brings together leading scientists, ethicists and public policy specialists to explore the field's potential benefits and risks, as well as ethical questions and regulatory issues. The new initiative launches with three grants totaling more than $1.6 million to The Hastings Center, the J. Craig Venter Institute, and the Woodrow Wilson International Center for Scholars.

 

"The Foundation has a long and rich tradition of funding scientific research," said Dr. Paul Joskow, President Alfred P. Sloan Foundation. "With synthetic biology, scientists have gone from reading to writing the genetic code; it's imperative that we take a carefully reasoned and systematic approach to understanding the full spectrum of ethical and policy issues that may arise as research and applications in this field develop."

 

At the Hastings Center (http://www.thehastingscenter.org/), Foundation funding will allow for in-depth investigation into ethical issues that may arise in connection with developments in synthetic biology. The project aims to make serious contributions to scholarly literature, produce a base for further scholarship, and inform public policymaking.

 

Alfred P. Sloan Foundation funding will allow the J. Craig Venter Institute (http://www.jcvi.org/) to examine potential societal concerns associated with developments in synthetic genomics. The project will both inform the scientific community about these issues while also educating the policy and journalistic communities about the science. As a result, scientists, journalists and policymakers will be able to engage in informed discussions.

 

A grant to the Woodrow Wilson International Center for Scholars (http://www.wilsoncenter.org/) will analyze evolving public perceptions of potential societal risks that may arise related to research in and applications of synthetic biology, clarify whether our existing regulatory systems can address relevant risks that may be associated with the science, and inform and educate policymakers.

 

"This program builds on the Foundation's biosecurity work and will establish a community of scientists, ethicists and policy specialists who can work synergistically on these issues," said Paula Olsiewski, Program Director, Alfred P. Sloan Foundation. "Ethical and policy discussions must be informed by the realities of the science and similarly the science must take into consideration societal concerns so that synthetic biology can be applied both inventively and wisely."

 

About the Alfred P. Sloan Foundation

The Alfred P. Sloan Foundation, established in 1934, makes grants to support original research and broad-based education related to science, technology, and economic performance; and to improve the quality of American life. The Foundation believes that a carefully reasoned and systematic understanding of the forces of nature and society, when applied inventively and wisely, can lead to a better world for all. Please visit the Foundation's Web site at www.sloan.org. 

Research Teams at J. Craig Venter Institute and Ludwig Institute for Cancer Research Uncover New Chromosomal Alterations in Cancer Using Transcriptome

ROCKVILLE, MD and NEW YORK, NY—January 27, 2009— Researchers from the J. Craig Venter Institute (JCVI) and the Ludwig Institute for Cancer Research (LICR) have uncovered new genomic alterations that lead to gene fusions in a breast cancer cell line by using 454 Life Sciences sequencing technology. The work, led by Qi Zhao of JCVI and Otavia L. Caballero, of LICR, is being published the week of January 26 in the early online edition of the Journal of the Proceedings of the National Academy of Sciences (PNAS).

 

Previous studies have shown that gene fusions are key gene alteration events in the development and progression of many kinds of cancers. The discovery of the best known gene fusion, BCR-ABL, led to the development of Gleevec® for the treatment of chronic myelogenous leukemia and other cancers.

 

In this proof of concept study the researchers focused on the transcriptome, a subset of genes in the genome that code for proteins. It has long been known that cancers arise from various types of genomic changes in certain cells. Continued advances and cost efficiencies of next generation DNA sequencing technologies are enabling this more precise and detailed examination of changes in the human genome that could be directly involved in cancer.

 

The JCVI/LICR researchers began with a well-characterized breast cancer cell line, HCC1954 and performed high-throughput transcriptome sequencing. Previous studies on this cell line have uncovered certain types of genetic mutations and chromosomal abnormalities associated with breast cancer. By conducting the in-depth transcript sequencing in this study and comparing these data to the previous studies a clearer picture is emerging of all the expressed genes some of which present in altered forms in the cancer cell line.

 

The team began by generating more than half a million 454 reads of cDNA sequences. After extensive data mining, the team uncovered 496 sequences that indicate chromosomal translocations. Of these 496, the team characterized 208 as inter-chromosomal abnormalities and 210 were intra-chromosomal abnormalities. From here the team performed more detailed validation experiments with a control cell line (HCC1954 BL).

 

Through further analysis the team confirmed six inter-chromosomal changes and one intra-chromosomal change that have the potential to affect the protein producing ability of at least nine genes. The researchers also discovered that chromosome 8 in the cancer cell line seemed to be very involved in some of the genomic rearrangements. This data confirms earlier studies showing that genomic instability in this area is implicated in breast and prostate cancers.

 

Most genes involved in the discovered chromosomal rearrangement events in this study have been implicated in cancers, such as the MRE11A protein that is associated with mutations in many types of tumors including in breast cancer. The team also identified the SAMD12 gene as being involved in both inter- and intra-chromosomal rearrangements. While not previously thought to play a role in the development of cancer, this study showed that this gene might be implicated in cancer.

 

The team concluded that transcriptome sequencing with next generation sequencing technologies such as the 454 Life Sciences platform is very adept at finding genomic rearrangements and mutations associated with cancers. With deeper sequencing coverage this approach could be a powerful and efficient way to discover all events associated with expressed genes including gene fusions, somatic mutations and alternative trans-splicing that lead to the development of cancer.

 

Robert Strausberg, Ph.D., Deputy Director of the JCVI and leader of the Human Genomic Medicine team noted, “This approach reveals alterations in the cancer genome within the active genes of cancer cells. Through the comparison with related normal cells we can glean those that are specific to cancer cells, thereby revealing their unique biology, as well as suggesting new approaches to detection, diagnosis and treatment of cancers.”

 

According to Andrew Simpson, Ph.D., Scientific Director of the LICR, “These studies are an important component of the Hilton-Ludwig Cancer Metastasis Initiative, focused on preventing and treating cancer metastasis. This program brings together interdisciplinary teams of expert scientists, working together to improve the lives of cancer patients. The current study represents one aspect of our teams’ creative approach in revealing previously unknown features of cancer that together will provide a platform for cancer prevention and intervention.”

 

About the J. Craig Venter Institute

 

The JCVI is a not-for-profit research institute in Rockville, MD and La Jolla, CA dedicated to the advancement of the science of genomics; the understanding of its implications for society; and communication of those results to the scientific community, the public, and policymakers. Founded by J. Craig Venter, Ph.D., the JCVI is home to approximately 400 scientists and staff with expertise in human and evolutionary biology, genetics, bioinformatics/informatics, information technology, high-throughput DNA sequencing, genomic and environmental policy research, and public education in science and science policy. The legacy organizations of the JCVI are: The Institute for Genomic Research (TIGR), The Center for the Advancement of Genomics (TCAG), the Institute for Biological Energy Alternatives (IBEA), the Joint Technology Center (JTC), and the J. Craig Venter Science Foundation. The JCVI is a 501 (c) (3) organization. For additional information, please visit http://www.JCVI.org.

 

About Ludwig Institute for Cancer Research

 

The Ludwig Institute for Cancer Research (LICR) is the largest international academic institute dedicated to understanding and controlling cancer. With nine Branches in seven countries, and numerous Affiliates and Clinical Trial Centers in many others, the scientific network that is LICR quite literally covers the globe. The uniqueness of LICR lies not only in its size and scale, but also in its philosophy and ability to drive its results from the laboratory into the clinic. LICR has developed an impressive portfolio of reagents, knowledge, expertise, and intellectual property, and has also assembled the personnel, facilities, and practices necessary to patent, clinically evaluate, license, and thus translate, the most promising aspects of its own laboratory research into cancer therapies.

Chloroplast membrane

Chloroplasts contain several important membranes, vital for their function. Like mitochondria, chloroplasts have a double-membrane envelope, called the chloroplast envelope. Each membrane is a phospholipid bilayer, between 6 and 8 nm thick, and the two are separated by a gap of 10-20nm, called the intermembrane space. The outer membrane is permeable to most ions and metabolites, but the inner membrane is highly specialised with transport proteins.

The origin of chloroplasts is now largely accepted by the botany community as occurring via endosymbiosis on an ancestral basis with the engulfment of photosynthetic bacterium within the eukaryotic cell. Over millions of years the endosymbiotic cyanobacterium evolved structurally and functionally, retaining its own DNA and cellular mitosis capabilities but losing its ablility to live outside of the host cell.

[edit]Internal parts

Within the inner membrane, in the region called the stroma, there is a system of interconnecting flattened membrane compartments, called thethylakoids. These are the sites of light absorption and ATP synthesis, and contain many proteins, including those involved in the electron transport chain. Photosynthetic pigments such as chlorophyll α and B, and some others e.g. xanthophylls and carotenoids are also located within this space. These are responsible for the conversion of light energy to chemical energy as described below:

[edit]Functions of Thylakoids

The membranes of the chloroplasts contain photosystems I and II which harvest solar energy in order to excite electrons which travel down theelectron transport chain. This exergonic fall in potential energy along the way is used to pump H+ ions from the stroma into the thylakoid space. A concentration gradient is formed, which allows chemiosmosis to occur, where the protein ATP synthase harvests the potential energy of the Hydrogen ions and uses it to combine ADP and a phosphate group to form ATP.

Experiments have shown that the pH within the stroma is about 7.8, while that of the thylakoid space is about 5. This corresponds to a thousandfold difference in concentration of H- ions.

Chloroplast

The inside of a chloroplast

Chloroplasts are organelles found in plant cells and eukaryotic algae that conductphotosynthesis. Chloroplasts absorb light and use it in conjunction with water and carbon dioxide to produce sugars, the raw material for energy and biomass production in all green plants and the animals that depend on them, directly or indirectly, for food. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis. The word chloroplast is derived from the Greek words chloros which means green andplast which means form or entity. Chloroplasts are members of a class of organelles known as plastids.

Evolutionary origin

Plant cells with visible chloroplasts.

Chloroplasts are one of the many different types of organelles in the cell. They are generally considered to have originated as endosymbiotic cyanobacteria (i.e. blue-green algae). This was first suggested by Mereschkowsky in 1905 ^[1] after an observation by Schimper in 1883 that chloroplasts closely resemble cyanobacteria. ^[2] All chloroplasts are thought to derive directly or indirectly from a single endosymbiotic event (in the Archaeplastida), except for Paulinellachromatophora, which has recently acquired a photosynthetic cyanobacterial endosymbiont which is not closely related to chloroplasts of other eukaryotes.^[3] In that they derive from an endosymbiotic event, chloroplasts are similar to mitochondria but chloroplasts are found only inplants and protista. The chloroplast is surrounded by a double-layered composite membrane with an intermembrane space; further, it has reticulations, or many infoldings, filling the inner spaces. The chloroplast has its own DNA which codes for redox proteins involved in electron transport in photosynthesis.

In green plants, chloroplasts are surrounded by two lipid-bilayer membranes. The inner membrane is now believed to correspond to the outer membrane of the ancestral cyanobacterium. Chloroplasts have their own genome, which is considerably reduced compared to that of free-living cyanobacteria, but the parts that are still present show clear similarities with the cyanobacterial genome. Plastids may contain 60-100 genes whereas cyanobacteria often contain more than 1500 genes.^[4] Many of the missing genes are encoded in the nuclear genome of the host. The transfer of nuclear information has been estimated in tobacco plants at one gene for every 16000 pollen grains.^[5]

In some algae (such as the heterokonts and other protists such as Euglenozoa and Cercozoa), chloroplasts seem to have evolved through a secondary event of endosymbiosis, in which a eukaryotic cell engulfed a second eukaryotic cell containing chloroplasts, forming chloroplasts with three or four membrane layers. In some cases, such secondary endosymbionts may have themselves been engulfed by still other eukaryotes, thus forming tertiary endosymbionts. In the alga Chlorella, there is only one chloroplast, which is bell shaped.

In some groups of mixotrophic protists such as the dinoflagellates, chloroplasts are separated from a captured alga or diatom and used temporarily. These klepto chloroplasts may only have a lifetime of a few days and are then replaced.^[6]

Structure

Chloroplasts are observable morphologically as flat discs usually 2 to 10 micrometer in diameter and 1 micrometer thick. In land plants they are generally 5 μm in diameter and 2.3 μm thick. The chloroplast is contained by an envelope that consists of an inner and an outer phospholipid membrane. Between these two layers is the intermembrane space. A typical parenchyma cell contains about 10 to 100 chloroplasts.

The material within the chloroplast is called the stroma, corresponding to the cytosol of the original bacterium, and contains one or more molecules of small circular DNA. It also contains ribosomes, although most of its proteins are encoded by genes contained in the host cell nucleus, with the protein products transported to the chloroplast.

Chloroplast ultrastructure:
1. outer membrane
2. intermembrane space
3. inner membrane (1+2+3: envelope)
4. stroma (aqueous fluid)
5. thylakoid lumen (inside of thylakoid)
6. thylakoid membrane
7. granum (stack of thylakoids)
8. thylakoid (lamella)
9. starch
10. ribosome
11. plastidial DNA
12. plastoglobule (drop of lipids)

Within the stroma are stacks of thylakoids, the sub-organelles which are the site of photosynthesis. The thylakoids are arranged in stacks called grana (singular: granum).^[7] A thylakoid has a flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes place on the thylakoid membrane; as in mitochondrial oxidative phosphorylation, it involves the coupling of cross-membrane fluxes withbiosynthesis via the dissipation of a proton electrochemical gradient.

In the electron microscope, thylakoid membranes appear as alternating light-and-dark bands, each 0.01 μm thick. Embedded in the thylakoid membrane is the antenna complex, which consists of the light-absorbing pigments, including chlorophyll and carotenoids, and proteins (which bind the chlorophyll). This complex both increases the surface area for light capture, and allows capture of photons with a wider range of wavelengths. The energy of the incident photons is absorbed by the pigments and funneled to the reaction centre of this complex through resonance energy transfer. Two chlorophyll molecules are then ionised, producing an excited electron which then passes onto the photochemical reaction centre.

Recent studies have shown that chloroplasts can be interconnected by tubular bridges called stromules, formed as extensions of their outer membranes.^[8][9] Chloroplasts appear to be able to exchange proteins via stromules,^[10] and thus function as a network.

Transplastomic plants

Recently, chloroplasts have caught attention by developers of genetically modified plants. In most flowering plants, chloroplasts are not inherited from the male parent, although in plants such as pines, chloroplasts are inherited from males.^[13] Where chloroplasts are inherited only from the female, transgenes in these plastids cannot be disseminated by pollen. This makes plastid transformation a valuable tool for the creation and cultivation of genetically modified plants that are biologically contained, thus posing significantly lower environmental risks. This biological containment strategy is therefore suitable for establishing the coexistence of conventional and organic agriculture. The reliability of this mechanism has not yet been studied for all relevant crop species. However, the research programme Co-Extra recently published results for tobacco plants, demonstrating that the containment of transplastomic plants is highly reliable with a tiny failure rate of 3 in 1,000,000.

Intermembrane space

Simplified structure of mitochondrion

Chloroplast

The intermembrane space is the region between the inner membrane and theouter membrane of a mitochondrion or a chloroplast. The main function of the intermembrane space is oxidative phosphorylation.

Channel proteins called porins in the outer membrane allow free movement of ionsand small molecules into the intermembrane space. This means that it is essentially continuous with the cytosol in terms of the solutes relevant for the functioning of these organelles. Enzymes destined for the mitochondrial matrix or the stroma can pass through the intermembrane space via transport throughtranslocators. These are known as translocase of the outer mitochondria membrane (TOM) and translocase of the inner mitochondrial membrane (TIM) in mitochondriaand translocase of the outer chloroplast membrane (TOC) and translocase of the inner chloroplast membrane (TIC) in chloroplasts. It tends to have a low pH because of the proton gradient which results when protons are pumped from themitochondrial matrix into the intermembrane space during electron transport. The structures responsible for this are coenzyme Q, NADH coenzyme Q oxidoreductasecomplex (complex I), succinate-coenzyme Q oxidoreductase complex (complex II), and coenzyme Q-cytochrome c oxidoreductase complex (complex III).

[edit]Intermembrane space of mitochondria

Main article: Intermembrane space of mitochondria

Because of channels in the outer membrane of the mitochondria, the content of the intermembrane space is similar to that of the content of the cytoplasm.

[edit]Intermembrane space of chloroplast

The intermembrane space of the chloroplast is extremely small, somewhere from 10-20 nm thick.

Mitochondrion

Electron micrograph of a mitochondrion from mammalian lung tissue showing its matrix and membranes.

In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells.^[1] These organelles range from 1–10 micrometers (μm) in size. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply ofadenosine triphosphate (ATP), used as a source of chemical energy. ^[2] In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth.^[3] Mitochondria have been implicated in several human diseases, including mitochondrial disorders^[4] and cardiac dysfunction,^[5] and may play a role in the aging process. The word mitochondrion comes from the Greek μίτος or mitos, thread + χονδρίον orkhondrion, granule. Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria.^[6][7] The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissues and species. In human, 615 distinct types of proteins were identified from cardiac mitochondria;^[8] whereas in murinae (rats), 940 proteins encoded by distinct genes were reported.^[9] The mitochondrial proteome is thought to be dynamically regulated.^[10] Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

Chromosome

Diagram of a duplicated and condensed (metaphase) eukaryotic chromosome. (1)Chromatid - one of the two identical parts of the chromosome after S phase. (2) Centromere - the point where the two chromatids touch, and where the microtubules attach. (3) Short arm. (4) Long arm.

A chromosome is an organized structure of DNA and protein that is found in cells. A chromosome is a single piece of DNA that contains many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions. The word chromosome comes from the Greekχρῶμα (chroma, color) and σῶμα (soma, body) due to their property of being stained very strongly by some dyes. Chromosomes vary extensively between different organisms. The DNA molecule may be circular or linear, and can contain anything from 10,000 to 1,000,000,000^[1]nucleotides in length. Typically eukaryotic cells (cells with nuclei) have large linear chromosomes and prokaryotic cells (cells without defined nuclei) have smaller circular chromosomes, although there are many exceptions to this rule. Furthermore, cells may contain more than one type of chromosome; for example, mitochondria in most eukaryotes andchloroplasts in plants have their own small chromosomes. In eukaryotes, nuclear chromosomes are packaged by proteins into a condensed structure called chromatin. This allows the very long DNA molecules to fit into the cell nucleus. The structure of chromosomes and chromatin varies through the cell cycle. Chromosomes may exist as either duplicated or unduplicated—unduplicated chromosomes are single linear strands, whereas duplicated chromosomes (copied during synthesis phase) contain two copies joined by a centromere. Compaction of the duplicated chromosomes during mitosis and meiosis results in the classic four-arm structure (pictured to the right).

"Chromosome" is a rather loosely defined term. In prokaryotes, a small circular DNA molecule may be called either a plasmid or a small chromosome. These small circular genomes are also found in mitochondria and chloroplasts, reflecting their bacterial origins. The simplest chromosomes are found in viruses: these DNA or RNA molecules are short linear or circular chromosomes that often lack any structural proteins.

Techniques of molecular biology

Since the late 1950s and early 1960s, molecular biologists have learned to characterize, isolate, and manipulate the molecular components of cells and organisms. These components include DNA, the repository of genetic information; RNA, a close relative of DNA whose functions range from serving as a temporary working copy of DNA to actual structural and enzymatic functions as well as a functional and structural part of the translational apparatus; and proteins, the major structural and enzymatic type of molecule in cells.

For more extensive list on protein methods, see protein methods.

For more extensive list on nucleic acid methods, see nucleic acid methods.

[edit]Expression cloning

Main article: Expression cloning

One of the most basic techniques of molecular biology to study protein function is expression cloning. In this technique, DNA coding for a protein of interest is cloned (using PCR and/or restriction enzymes) into a plasmid (known as an expression vector). This plasmid may have special promoter elements to drive production of the protein of interest, and may also have antibiotic resistance markers to help follow the plasmid.

This plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation (via uptake of naked DNA), conjugation (via cell-cell contact) or by transduction (via viral vector). Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection,electroporation, microinjection and liposome transfection. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers, the latter is sometimes called bactofection and in particular uses Agrobacterium tumefaciens. The plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection.

In either case, DNA coding for a protein of interest is now inside a cell, and the protein can now be expressed. A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can then be extracted from the bacterial or eukaryotic cell. The protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied.

[edit]Polymerase chain reaction (PCR)

Main article: Polymerase chain reaction

The polymerase chain reaction is an extremely versatile technique for copying DNA. In brief, PCR allows a single DNA sequence to be copied (millions of times), or altered in predetermined ways. For example, PCR can be used to introduce restriction enzyme sites, or to mutate (change) particular bases of DNA, the latter is a method referred to as "Quick change". PCR can also be used to determine whether a particular DNA fragment is found in a cDNA library. PCR has many variations, like reverse transcription PCR (RT-PCR) for amplification of RNA, and, more recently, real-time PCR (QPCR) which allow for quantitative measurement of DNA or RNA molecules.

[edit]Gel electrophoresis

Main article: Gel electrophoresis

Gel electrophoresis is one of the principal tools of molecular biology. The basic principle is that DNA, RNA, and proteins can all be separated by means of an electric field. In agarose gel electrophoresis, DNA and RNA can be separated on the basis of size by running the DNA through an agarose gel. Proteins can be separated on the basis of size by using an SDS-PAGE gel, or on the basis of size and their electric charge by using what is known as a 2D gel electrophoresis.

[edit]Southern blotting

Main article: Southern blot

Named after its inventor, biologist Edwin Southern, the Southern blot is a method for probing for the presence of a specific DNA sequence within a DNA sample. DNA samples before or after restriction enzyme digestion are separated by gel electrophoresis and then transferred to a membrane by blotting via capillary action. The membrane is then exposed to a labeled DNA probe that has a complement base sequence to the sequence on the DNA of interest. Most original protocols used radioactive labels, however non-radioactive alternatives are now available. Southern blotting is less commonly used in laboratory science due to the capacity of other techniques, such as PCR, to detect specific DNA sequences from DNA samples. These blots are still used for some applications, however, such as measuring transgene copy number intransgenic mice, or in the engineering of gene knockout embryonic stem cell lines.

[edit]Northern blotting

Main article: northern blot

The northern blot is used to study the expression patterns a specific type of RNA molecule as relative comparison among of a set of different samples of RNA. It is essentially a combination of denaturing RNA gel electrophoresis, and a blot. In this process RNA is separated based on size and is then transferred to a membrane that is then probed with a labeled complement of a sequence of interest. The results may be visualized through a variety of ways depending on the label used; however, most result in the revelation of bands representing the sizes of the RNA detected in sample. The intensity of these bands is related to the amount of the target RNA in the samples analyzed. The procedure is commonly used to study when and how much gene expression is occurring by measuring how much of that RNA is present in different samples. It is one of the most basic tools for determining at what time, and under what conditions, certain genes are expressed in living tissues.

[edit]Western blotting

Main article: western blot

Antibodies to most proteins can be created by injecting small amounts of the protein into an animal such as a mouse, rabbit, sheep, or donkey (polyclonal antibodies)or produced in cell culture (monoclonal antibodies). These antibodies can be used for a variety of analytical and preparative techniques.

In western blotting, proteins are first separated by size, in a thin gel sandwiched between two glass plates in a technique known as SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis). The proteins in the gel are then transferred to a PVDF, nitrocellulose, nylon or other support membrane. This membrane can then be probed with solutions of antibodies. Antibodies that specifically bind to the protein of interest can then be visualized by a variety of techniques, including colored products, chemiluminescence, or autoradiography. Often, the antibodies are labeled with an enzymes. When a chemiluminescent substrate is exposed to the enzyme it allows detection. Using western blotting techniques allows not only detection but also quantitative analysis.

Analogous methods to western blotting can be used to directly stain specific proteins in live cells or tissue sections. However, theseimmunostaining methods, such as FISH, are used more often in cell biology research.

[edit]Blotting jokes

The terms "western" and "northern" are molecular biology jokes that play on the term southern blot. The first blots were with DNA, and since they were done by Ed Southern, they came to be known as Southerns. Patricia Thomas, inventor of the RNA blot, which became known as a "northern", actually didn't use the term. ^[2]. To carry the joke further, one can find references in the literature to "southwesterns" (protein-DNA interactions), "northwesterns" (protein-RNA interactions) and "farwesterns" (protein-protein interactions).

[edit]Arrays

Main article: DNA microarray

A DNA array is a collection of spots attached to a solid support such as a microscope slide where each spot contains one or more single-stranded DNA oligonucleotide fragment. Arrays make it possible to put down a large quantity of very small (100 micrometre diameter) spots on a single slide. Each spot has a DNA fragment molecule that is complementary to a single DNA sequence (similar to Southern blotting). A variation of this technique allows the gene expression of an organism at a particular stage in development to be qualified (expression profiling). In this technique the RNA in a tissue is isolated and converted to labeled cDNA. This cDNA is then hybridized to the fragments on the array and visualization of the hybridization can be done. Since multiple arrays can be made with the exact same position of fragments they are particularly useful for comparing the gene expression of two different tissues, such as a healthy and cancerous tissue. Also, one can measure what genes are expressed and how that expression changes with time or with other factors. For instance, the common baker's yeast,Saccharomyces cerevisiae, contains about 7000 genes; with a microarray, one can measure qualitatively how each gene is expressed, and how that expression changes, for example, with a change in temperature. There are many different ways to fabricate microarrays; the most common are silicon chips, microscope slides with spots of ~ 100 micrometre diameter, custom arrays, and arrays with larger spots on porous membranes (macroarrays). There can be anywhere from 100 spots to more than 10,000 on a given array.

Arrays can also be made with molecules other than DNA. For example, an antibody array can be used to determine what proteins or bacteriaare present in a blood sample.

[edit]Allele Specific Oligonucleotide

Allele specific oligonucleotide (ASO) is a technique that allows detection of single base mutations without the need for PCR or gel electrophoresis. Short (20-25 nucleotides in length), labeled probes are exposed to the non-fragmented target DNA. Hybridization occurs with high specificity due to the short length of the probes and even a single base change will hinder hybridization. The target DNA is then washed and the labeled probes that didn't hybridize are removed. The target DNA is then analyzed for the presence of the probe via radioactivity or fluorescence. In this experiment, as in most molecular biology techniques, a control must be used to ensure successful experimentation.

[edit]Abandoned technology

As new procedures and technology become available, the older technology is rapidly abandoned. A good example is methods for determining the size of DNA molecules. Prior to gel electrophoresis (agarose or polyacrylamide) DNA was sized with rate sedimentation in sucrose gradients, a slow and labor intensive technology requiring expensive instrumentation; prior to sucrose gradients, viscometry was used.

Aside from their historical interest, it is worth knowing about older technology as it may be useful to solve a particular problem.