Restriction Endonucleases: An Overview

restriction enzyme30 years ago, when people are restrictions on the phage host specificity – when modified to study the phenomenon , first discovered by restriction endonucleases . Bacteria can resist new viruses , and this ” limitation ” approach can be attributed to the survival of the virus inside the cell can destroy foreign DNA restriction endonucleases . The first restriction endonuclease from Escherichia coli is found to include the EcoR I and EcoR II, from Heamophilus influenzae and the Hind II and Hind III. These enzymes can cut DNA at specific sites can be generated in vitro ligation of the gene fragments . Researchers soon discovered endonuclease gene is composed of function and expression are very useful tools.

When the restriction endonuclease used in the seventies , when spread to many companies to start looking NEB represented within more restriction enzymes . Except for certain viruses, restriction endonuclease is found only in prokaryotes . People are looking for the new restriction endonuclease from thousands of bacteria and archaea . And prokaryotic genome sequenced data analysis showed that the restriction enzymes ubiquitous in prokaryotes – free survival of all bacteria and archaea can seem encoding restriction endonuclease .

Restriction endonuclease forms, from the size of it , they can be as small as Pvu II (157 amino acids ) can also be more than 1250 amino acids Cje I greater. In the 3000 classification purified restriction enzymes , it has been found more than 250 kinds of specific recognition sequence. Of which 30% are found in NEB . With unknown specificity of restriction endonuclease recognition sequence found work continues. People from the perspective of the analysis of biochemical study of cell extracts , but also known by computer analysis of genomic data in order to discover more . Although many of the newly discovered enzyme recognition sequence has been repeated with – that isoschizomers , there is still a new recognition sites for the enzyme being discovered .

In the 1980s , NEB began cloning and expression of restriction endonucleases . Since the cloning of the expression of the restriction endonuclease and separated from the original environment of the cell , to avoid contamination of other cells in the original enzyme , thereby enhancing the purity of the enzyme . Furthermore, to improve the cloning restriction endonuclease yields , simplifying the purification process , so that significantly reduce production costs ; easily cloned genes were sequenced , the protein is also analyzed by X -ray crystallography , which allows us to cloning product more certain .

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Isolation and characterization of two distinct cDNA clones encoding corn seed cysteine proteinases

Domoto, Chieko; Watanabe, Hirohito; Abe, Makoto; Abe, Keiko; Arai, Soichi, 1995: Isolation and characterization of two distinct cDNA clones encoding corn seed cysteine proteinases. Biochimica Et Biophysica Acta. 1263(3): 241-244

We obtained two cDNA clones encoding corn seed cysteine proteinases (Ccp1 and Ccp2). Sequence analysis showed that Ccp1 is made of 371 protein residues, in the prepro-protein form, with two unique short insertions from the mature protein region who are not within papain or other common CPs. Ccp2 contains 360 protein residues with a vacuole sorting signal inside the pro-sequence region. An protein sequence similarity of 42% was found between your mature protein region of Ccp1 and that of Ccp Although Ccp1 is very homologous to pea 15a Cp (72%) and Arabidopsis thaliana Rd19 Cp (79%), because both versions are known to be induced not until guarana is exposed to a dehydrated environment, it showed really low homologies with known cysteine proteinases (38-43%). Ccp2 showed around 87% and 89% identity to rice oryzain gamma and barley aleurain, respectively. We also observed that this Ccp1 mRNA is expressed in ripened corn seeds, although its expression reaches a maximum 5 days as soon as the oncoming of germination. On the other hand, the Ccp2 mRNA is expressed only during germination, with maximum expression on the 3-day stage. These results suggest arsenic intoxication no less than two cysteine proteinases playing differential roles inside the corn seeds.

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This Week in PLOS NTDs and PLOS Pathogens: Climate, Chemicals and Vector Control, the Incubation of Buruli Ulcer, Supressing Viral Growth, and More By PLOS Neglected Tropical Diseases and PLOS Pathogens

Buruli ulcer’s mode of transmission is presently unknown, but in Victoria, Australia, endemic areas have been carefully mapped and patients are known to report single visits or defined exposures to these areas. From a retrospective review, Jason Trubiano and colleagues identified 23 patients with a single visit exposure to BU-endemic regions from a total of 408 diagnoses in Victoria over a decade and have estimated a likely incubation period of Mycobacterium ulcerans.

In this paper, Julien Lemaire and colleagues evaluate the impact of Leishmania major infection on deregulation of non-coding miRNAs, a class of important regulators of gene expression. Their results reveal the implication of several miRNAs on macrophage fate upon parasite infection through regulation of different pathways including cell death. These findings provide new insight for understanding mechanisms governing this miRNA deregulation by parasite infection and will help to provide clues for the development of control strategies for leishmaniasis.

It has been unknown how Leptospira cause disease and why different strains cause different severity of illness. In this study Jason Lehmann and colleagues attenuated a highly virulent strain of L. interrogans by culturing it in vitro over several months. Comparison of the whole genome sequence before and after the attenuation process revealed a small set of genes that were mutated, and therefore associated with virulence — a result that aids in the understanding of Leptospira evolution and pathogenesis.

The following new articles are publishing in PLOS Pathogens this week:

Chemicals are powerful tools in the control of malaria and other vector-borne diseases. Because temperature can affect the activity of chemicals used for vector control, Krijn P. Paaijmans and colleagues argue that candidate chemicals need to be evaluated under relevant climatic conditions. They also suggest that the range of temperatures currently recommended for development and field testing of new chemicals should be expanded.

Little is known about the early stages of primary pneumonic plague caused by pulmonary exposure to Yersinia pestis. Working with fully virulent Y. pestis in mice, William Goldman and colleagues now report that the bacteria primarily target pulmonary macrophages and neutrophils during the pre-inflammatory phase of disease, and that neutrophils are responsible for the severe necrotizing pneumonia during the pro-inflammatory disease phase.

Screening for substances that stimulate expression of interferon-inducible antiviral genes, Hélène Munier-Lehmann, Frédéric Tangy, Pierre-Olivier Vidalain and colleagues isolated compound DD246 from a chemical library and found that it targets the pyrimidine biosynthesis pathway. Their study establishes a link between inhibition of pyrimidine biosynthesis, amplification of antiviral gene expression, and inhibition of RNA virus infections.

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Cellecta’s DECIPHER Project RNAi Screening Tools at Roswell Park Cancer Institute

We’ve started your blog as being a simple, unobtrusive conduit that permits us to speak on to our customers, collaborators, along with interested researchers about new and interesting developments linked to Cellecta’s technology and business. Because the first post, we thought i would let you know about our recent agreement using the Roswell Park Cancer Institute (RPCI) that delivers support to laboratories in their institution doing genome-wide RNAi screenings using our expanding portfolio of open-access DECIPHER™ pooled lentiviral shRNA expression libraries.

However the plasmid versions in the DECIPHER shRNA library “modules” can be obtained free-of-charge to any academic laboratory though the DECIPHER Project, the DECIPHER Technology Access and Maintenance (TAM) Program, that your RPCI just joined, lets them choose this screening technology accessible by using a well-supported centralized core facility. The DECIPHER TAM Program enables us to deliver this institution with new modules of the packaged, ready-to-use shRNA expression libraries as they are developed, proactive support for those labs using these resources, updates on new technological developments and protocols, and access to the most up-to-date software to handle screening results.

Currently, as part of the open-access DECIPHER Project, you will discover 4 pooled shRNA library modules freely available—two modules targeting 10,000 human genes and also targeting 10,000 mouse genes. Each one library module targets approximately 5,000 well-annotated genes with 27,500 shRNAs (5-6 shRNA target each transcript). Human and mouse 1 modules target that same pair of genes relevant to signal transduction and cancer. Your second modules target a broader variety of genes that seem to be linked to disease processes and pathology but weren’t targeted in the first module. In some weeks, we will to discharge human module 3 targeting 5,000 genes. Ultimately, you will have 5 modules targeting all human genes. Much like all of our libraries, we utilize bar-coded inserts and have absolutely checked the products the DECIPHER modules using HT sequencing to make certain all shRNA sequences exist at sufficient levels to make certain comprehensive screening from the targeted gene set.

A chance to identify genes with specific functional activities makes screening with pooled shRNA expression libraries a very powerful technique to investigate the genetic controls regulating a variety of biological responses. However, effective screening might be technically challenging. As the DECIPHER Project helps to fulfill our goal to make basic tools due to this form of analysis easily accessible to all or any labs, the DECIPHER TAM Program allows us to provide you with the resources and support that could empower labs to make use of these RNAi screening tools to their maximum potential. We are really awaiting utilizing RPCI about this program.

More information around the DECIPHER Project can be located on http://www.decipherproject.net.

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Technique Enables Hands-free Heart, Respiration Measurements

A fairly easy video camera followed by complex algorithms seems to be provide an accurate ways to remotely monitor heart and respiration rates night or day, researchers report.

The inexpensive way of monitoring the vital signs without touching a person could have major implications for telemedicine, including enabling rapid detection of an cardiac event or stroke occurring both at home and helping avoid crib death, based on a survey published from the journal PLOS ONE.

It also may enable untethered, more realistic monitoring of laboratory animals in research project together with ecological research on wild or endangered animal populations.

“Heart and respiratory rates obviously tell us a great deal precisely someone does,” said Dr. Joe Tsien, neuroscientist on the Medical College of Georgia at Georgia Regents University. “Normally, caregivers ought to put their face to face an individual to assess these rates. However our algorithms enable us to rapidly and accurately translate, by way of example, normally imperceptible movement of the epidermis in rhythm with the breathing into a definative way of measuring respiration rate.”

Scientists at MCG and China’s BanNa Biomedical Research Institute are already accommodating check if the approach may accurately measure blood pressure level.

Tsien, who studies memory, said the algorithms were developed to decipher reams of info generated by his brain-decoding project, and that is identifying brain activity patterns that occur, one example is, each time a mouse forms a memory. Therefore, the formulas help him realize what as their pharmicudical counterpart is saying; in cases like this, help interpret how the person is doing.

To measure heart rate, this process takes good thing about the truth that the arteries expand and contract with each heartbeat: more blood inside vessels means more camera light is absorbed as an alternative to reflected. Breathing results in a slight body movement that produces varying lengths of reflected light over moving surface. In reality, Tsien notes, each rates are closely tied and respiration rate can also be calculated dependant on pulse, using his algorithms along with other methods.

In just a couple of seconds, the algorithms enable light reflections for being sorted by source to ensure ambient signals from, say, a fluorescent lamp, might be ignored and distinct numbers might be calculated for heart and respiration rates.

“It lets us retrieve the faint but relevant signal that may be buried under ambient distractions,” Tsien said.

False-positive rates were lower than 3 % and false negatives under 1 percent, indicating the device could well be reliable even just in changing fast scenarios for example a stroke or stroke.

Measurements were taken several times on 15 human subjects, including seven males, eight females and something infant, who were a variety of Caucasians, Asians and blacks. To assess accuracy, heart and respiratory rates were concurrently measured using standard approaches for example an electrocardiogram for your heart and airflow captured by way of sensor under the nose while subjects were active and stationary. Similar studies were performed on mice, pigs and zebrafish.

To help promote assess accuracy, scientists also used the approach on still images for example photographs of humans, a Simpson childrens favourite and also the Birth of venus painting. Their system correctly identified all as  inanimate objects. In addition , they applied the strategy to television footage of celebrities for instance Michael Phelps and President Bill Clinton, and had the ability to detect varying heart and respiration rates in calm, happy and stressful situations.

Although researchers used an individual-channel camera – which produces images from a single source of light – their algorithm can work with any camcorder and also other sensors, for example radio frequency waves, Tsien said. However, the researchers realize that a previous attempt by others utilizing a multi-channel camera for remote monitoring didn’t accurately measure parameters in the evening and generated now more false positives.

Tsien is the Georgia Research Alliance Eminent Scholar in Cognitive and Systems Neurobiology. GRU Postdoctoral Fellow Fang Zhao will be the study’s first author.  GRU has patented the monitoring technique.

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New technique in RNA interference cuts a serious amounts of cost in genetic screens

There exists a new contender in neuro-scientific gene discovery, and yes it’s giving knockout mice a run their money. Researchers for the Rockefeller University have shown which a new technique using RNA interference has the capacity to find genes that create epidermal tumor increase in months rather than the decades it could take using traditional methods employing specially bred, genetically altered mice. They recently revealed the first genome-wide RNA interference screen of an mouse in the article published naturally.

RNA interference is a natural process where RNA molecules inhibit gene expression, nonetheless it can also be used by scientists to dam a gene’s function to check out those that give rise to certain diseases.

“For decades, fruit flies and worms are already great model organisms due to the capability to accomplish rapid genetic screens,” says Elaine Fuchs, Rebecca C. Lancefield Professor. “Genetic screens in mammalian cells happen to be restricted to petri dishes, where cells experience stress and nonphysiological growth conditions.”

Doing the screen involves using short waste RNA, called small hairpin RNAs, which can be inserted into the cell and are capable of halt messages from specific genes, keeping the genes from making proteins. A genetic screen might consider 15,000 genes — meaning 1000s of petri dishes or fruit flies — a task deemed far too large, time-consuming and expensive for do with current mouse knockout technology. But researchers led by Slobodan Beronja, an ancient postdoc in Fuchs’s Laboratory of Mammalian Cell Biology and Development, created a special technique of RNA interference, the place that the small hairpin RNAs are pooled together and injected into your embryos of pregnant mice by using a virus.

“We’ve now devised a technology where we can easily effectively treat the top of living mouse embryos as a petri dish of cells, and accomplish genome wide screens on mouse cells into their native environment in vivo,” says Fuchs. “Previously, genome-wide screens were only possible in lower animals, for instance flies, worms and yeast, where it is often hard to look at the relevance to human disorders for example cancer.”

After allowing for normal or pre-cancerous tissues to grow, the researchers quantified the volume of individual small hairpin RNAs within the animals, and put on the extender like a measure of their relative importance on the growth process, Beronja, now an assistant member on the Fred Hutchinson Cancer Research facility, explains. The results could be the chance to screen more genes with fewer mice. The c’s screened over 16,000 genes using just 100 litters of mice, and identified about 200 genes which are uniquely important to oncogenic growth in your skin.

“We needed to identify new genes which have been worth creating drugs against in cancer treatment,” says Beronja. “The higher the complexness with the medications, the more its success. Should you attack several genes, it’s more potent.”

They found several genes that had been already known to cause tumor growth, but a majority of, including one generally known as Mllt6, surprised them. Nevertheless , there has become some study about it in leukemia, the gene had never been connected to solid tumors.

“The next thing is to validate the list of 200-plus genes and narrow it as a result of the truth candidates for drug therapy. Mllt6 is but one that needs further exploration,” says Beronja. “But we have shown until this method of genetic screening will be less, easier and more informative. It absolutely was a risky undertaking that Dr. Fuchs entrusted me for taking, and it also paid.”

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Measles’ Natural Nemesis: Cells Infected by Measles Virus Pull out a Heavy Weapon in the Form of the Enzyme

Scientists at The Scripps Research Institute have found that a known enzyme in cells protects against measles virus, likely by altering the virus’s genetic material, RNA. Cells lacking the enzyme become highly vulnerable to the virus’s destructive effects. The enzyme also protects against several other respiratory viruses, including influenza A.

“We believe that host cells use this RNA-editing enzyme to slow these viruses’ ability to replicate,” said Michael B. A. Oldstone, the study’s senior author and a professor at Scripps Research’s La Jolla, California campus. The study’s first authors are Simone V. Ward, a senior research associate in the Scripps Research Oldstone laboratory, and Cyril X. George of the University of California, Santa Barbara.

The finding represents a significant improvement in the understanding of measles infections, which still kill about 150,000 children and adults around the world every year. The paper, which was published recently in Proceedings of the National Academy of Sciences, has prompted commentaries in the journals Nature Reviews Microbiology and Viruses.

The focus of the study was the enzyme ADAR1 (“adenosine deaminase acting on RNA, 1”), which is known to be produced in high amounts in measles-infected cells. ADAR1 has been suspected as a “restriction factor” that inhibits viral replication.

ADAR1’s role against measles has been difficult to nail down, however. In mice genetically engineered to be infectable by measles — a virus that normally infects only humans — ADAR1 is required for embryonic development, as in all mice. Thus the standard “gene knockout” technique, which would enable scientists to see how measles infections proceed without ADAR1, hasn’t been feasible.

In this study, Ward, George, and Oldstone, and their colleagues knocked out only one of the two forms of ADAR1 produced in cells. This form, p150, is the one produced in response to infections. For reasons that still aren’t clear, mouse embryos cannot grow for long without p150, so the researchers used a standard technique to “immortalize” these p150-knockout embryonic cells — ensuring their continuous supply — and in this way created a useful cell model.

When infected by measles virus, the p150-knockout cells succumbed quickly compared to immortalized control cells that produced p150 normally. “When I looked at the cells only 21 hours after infection, the p150-knockout cells already showed the signs of cell damage typical of measles infection,” said Ward. “But the control cells looked exactly like uninfected cells.”

In further tests, Ward found p150 also provided significant protection for cells against Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus — which are all respiratory viruses like measles, and all members of the paramyxovirus or orthomyxovirus families.

Nine years ago, it was reported that a restriction enzyme known as APOBEC3G protects cells from DNA-based viruses such as HIV, by mutating viral genes. “We’re now showing that an analogous gene-editing enzyme also seems to exist for RNA viruses,” said Oldstone.

With the new cell model, and advanced “conditional knockout” techniques that allow genes to be disrupted in specific organs in adult mice, Ward, Oldstone, and their colleagues now hope to study ADAR1-p150’s role in more detail.

One key issue to be resolved is the enzyme’s role during brain infections. Measles virus usually results in a relatively mild illness lasting only a week or two, but in rare cases it spreads to the brain and becomes a persistent, always fatal infection known as subacute sclerosing panencephalitis (SSPE). In such cases, the virus doesn’t have to spread via cell-to-cell contact, thus exposing itself to the immune system; it can spread more stealthily along the axons and dendrites that connect neurons.

“What we hope to show with our ongoing work is that host neurons are using ADAR1 to slow down this process, turning it into a gradual neurological disease,” said Oldstone. ADAR1 might also be exacerbating the neurological symptoms of SSPE, he adds, because its enzymatic activity is known to affect the production of the important neurotransmitter receptors for serotonin and glutamate: “It’s an enzyme that has multiple roles,” Oldstone concluded.

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