Information

What is functional dissection?


Reading [1] I found the sentence:

Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts.

I did not find a definition online yet. What is the meaning of functional dissection in this context?

[1] K. Oshima et al., “Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia,” Proc Natl Acad Sci USA, vol. 113, no. 40, pp. 11306-11311, Oct. 2016.


The term 'functional' in this context implies a type of analysis that is able to provide a mechanistic understanding.

The authors first describe their exploratory approach, by which they identified some genes and their mutations that are associated with patient relapse in acute lymphoblastic leukemia. Then they proceed to intervene in the system, by expressing mutated KRAS in mice. When they did that, they observed several hallmarks of relapse in those mice. Hence they can conclude that a mutation of this gene is involved in the mechanism that underlies relapse.

Thus the way these authors use the term 'functional analysis'/'functional dissection' is in contrast to a correlational approach.


The underlying assumption is that by isolating parts of the system and studying their properties and their effects we will be able to understand the physical world. This is a philosophical (metaphysical) thesis called reductionism, which is arguably the prevailing view in the natural sciences.

The first use of the term "functional dissection" I could find is in a 1971 paper on the immunological response of small molecules. Their goal in that paper was to dissociate (or dissect) the effects of small molecules, such as a bovine glucagon, on antibody specificity versus antibody production. They have found that different parts of this molecule were responsible for antigen specificity (all antibodies recognised the N-terminal) and cellular immunity (mostly the C-terminal of the molecule was associated with DNA synthesis).


Functional disorder

A functional disorder is a medical condition that impairs normal functioning of bodily processes that remains largely undetected under examination, dissection or even under a microscope. At the exterior, there is no appearance of abnormality. This stands in contrast to a structural disorder (in which some part of the body can be seen to be abnormal) or a psychosomatic disorder (in which symptoms are caused by psychological or psychiatric illness). Definitions vary somewhat between fields of medicine.

Generally, the mechanism that causes a functional disorder is unknown, poorly understood, or occasionally unimportant for treatment purposes. The brain or nerves are often believed to be involved. It is common that a person with one functional disorder will have others.


Contents

Plant and animal bodies are dissected to analyze the structure and function of its components. Dissection is practised by students in courses of biology, botany, zoology, and veterinary science, and sometimes in arts studies. In medical schools, students dissect human cadavers to learn anatomy. [1]

Dissection is used to help to determine the cause of death in autopsy (called necropsy in other animals) and is an intrinsic part of forensic medicine. [2]

A key principle in the dissection of human cadavers is the prevention of human disease to the dissector. Prevention of transmission includes the wearing of protective gear, ensuring the environment is clean, dissection technique [3] and pre-dissection tests to specimens for the presence of HIV and hepatitis viruses. [4] Specimens are dissected in morgues or anatomy labs. When provided, they are evaluated for use as a "fresh" or "prepared" specimen. [4] A "fresh" specimen may be dissected within some days, retaining the characteristics of a living specimen, for the purposes of training. A "prepared" specimen may be preserved in solutions such as formalin and pre-dissected by an experienced anatomist, sometimes with the help of a diener. [4] This preparation is sometimes called prosection. [5]

Most dissection involves the careful isolation and removal of individual organs, called the Virchow technique. [3] [6] An alternative more cumbersome technique involves the removal of the entire organ body, called the Letulle technique. This technique allows a body to be sent to a funeral director without waiting for the sometimes time-consuming dissection of individual organs. [3] The Rokitansky method involves an in situ dissection of the organ block, and the technique of Ghon involves dissection of three separate blocks of organs - the thorax and cervical areas, gastrointestinal and abdominal organs, and urogenital organs. [3] [6] Dissection of individual organs involves accessing the area in which the organ is situated, and systematically removing the anatomical connections of that organ to its surroundings. For example, when removing the heart, connects such as the superior vena cava and inferior vena cava are separated. If pathological connections exist, such as a fibrous pericardium, then this may be deliberately dissected along with the organ. [3]

Classical antiquity Edit

Human dissections were carried out by the Greek physicians Herophilus of Chalcedon and Erasistratus of Chios in the early part of the third century BC. [7] During this period, the first exploration into full human anatomy was performed rather than a base knowledge gained from 'problem-solution' delving. [8] While there was a deep taboo in Greek culture concerning human dissection, there was at the time a strong push by the Ptolemaic government to build Alexandria into a hub of scientific study. [8] For a time, Roman law forbade dissection and autopsy of the human body, [9] so physicians had to use other cadavers. Galen, for example, dissected the Barbary macaque and other primates, assuming their anatomy was basically the same as that of humans. [10] [11] [12]

India Edit

The ancient societies that were rooted in India left behind artwork on how to kill animals during a hunt. [13] The images showing how to kill most effectively depending on the game being hunted relay an intimate knowledge of both external and internal anatomy as well as the relative importance of organs. [13] The knowledge was mostly gained through hunters preparing the recently captured prey. Once the roaming lifestyle was no longer necessary it was replaced in part by the civilization that formed in the Indus Valley. Unfortunately, there is little that remains from this time to indicate whether or not dissection occurred, the civilization was lost to the Aryan people migrating. [13]

Early in the history of India (2nd to 3rd century), the Arthashastra described the 4 ways that death can occur and their symptoms: drowning, hanging, strangling, or asphyxiation. [14] According to that source, an autopsy should be performed in any case of untimely demise. [14]

The practice of dissection flourished during the 7th and 8th century. It was under their rule that medical education was standardized. This created a need to better understand human anatomy, so as to have educated surgeons. Dissection was limited by the religious taboo on cutting the human body. This changed the approach taken to accomplish the goal. The process involved the loosening of the tissues in streams of water before the outer layers were sloughed off with soft implements to reach the musculature. To perfect the technique of slicing, the prospective students used gourds and squash. These techniques of dissection gave rise to an advanced understanding of the anatomy and the enabled them to complete procedures used today, such as rhinoplasty. [13]

During medieval times the anatomical teachings from India spread throughout the known world however, the practice of dissection was stunted by Islam. [13] The practice of dissection at a university level was not seen again until 1827, when it was performed by the student Pandit Madhusudan Gupta. [13] Through the 1900s, the University teachers had to continually push against the social taboos of dissection, until around 1850 when the universities decided that it was more cost effective to train Indian doctors than bring them in from Britain. [13] Indian medical schools were, however, training female doctors well before those in England. [13]

The current state of dissection in India is deteriorating. The number of hours spent in dissection labs during medical school has decreased substantially over the last twenty years. [13] The future of anatomy education will probably be an elegant mix of traditional methods and integrative computer learning. [13] The use of dissection in early stages of medical training has been shown more effective in the retention of the intended information than their simulated counterparts. [13] However, there is use for the computer-generated experience as review in the later stages. [13] The combination of these methods are intended to strengthen the students understanding and confidence of anatomy, a subject that is infamously difficult to master. [13] There is a growing need for anatomist—seeing as most anatomy labs are taught by graduates hoping to complete degrees in anatomy—to continue the long tradition of anatomy education. [13]

Islamic world Edit

From the beginning of the Islamic faith in 610 A.D., [15] Shari'ah law has applied to a greater or lesser extent within Muslim countries, [15] supported by Islamic scholars such as Al-Ghazali. [16] Islamic physicians such as Ibn Zuhr (Avenzoar) (1091–1161) in Al-Andalus, [17] Saladin's physician Ibn Jumay during the 12th century, Abd el-Latif in Egypt c. 1200, [18] and Ibn al-Nafis in Syria and Egypt in the 13th century may have practiced dissection, [16] [19] [20] but it remains ambiguous whether or not human dissection was practiced. Ibn al-Nafis, a physician and Muslim jurist, suggested that the "precepts of Islamic law have discouraged us from the practice of dissection, along with whatever compassion is in our temperament", [4] indicating that while there was no law against it, it was nevertheless uncommon. Islam dictates that the body be buried as soon as possible, barring religious holidays, and that there be no other means of disposal such as cremation. [15] Prior to the 10th century, dissection was not performed on human cadavers. [15] The book Al-Tasrif, written by Al-Zahrawi in 1000 A.D., details surgical procedure that differed from the previous standards. [21] The book was an educational text of medicine and surgery which included detailed illustrations. [21] It was later translated and took the place of Avicenna's The Canon of Medicine as the primary teaching tool in Europe from the 12th century to the 17th century. [21] There were some that were willing to dissect humans up to the 12th century, for the sake of learning, after which it was forbidden. This attitude remained constant until 1952, when the Islamic School of Jurisprudence in Egypt ruled that "necessity permits the forbidden". [15] This decision allowed for the investigation of questionable deaths by autopsy. [15] In 1982, the decision was made by a fatwa that if it serves justice, autopsy is worth the disadvantages. [15] Though Islam now approves of autopsy, the Islamic public still disapproves. Autopsy is prevalent in most Muslim countries for medical and judicial purposes. [15] In Egypt it holds an important place within the judicial structure, and is taught at all the country's medical universities. [15] In Saudi Arabia, whose law is completely dictated by Shari'ah, autopsy is viewed poorly by the population but can be compelled in criminal cases [15] human dissection is sometimes found at university level. [15] Autopsy is performed for judicial purposes in Qatar and Tunisia. [15] Human dissection is present in the modern day Islamic world, but is rarely published on due to the religious and social stigma. [15]

Tibet Edit

Tibetan medicine developed a rather sophisticated knowledge of anatomy, acquired from long-standing experience with human dissection. Tibetans had adopted the practice of sky burial because of the country's hard ground, frozen for most of the year, and the lack of wood for cremation. A sky burial begins with a ritual dissection of the deceased, and is followed by the feeding of the parts to vultures on the hill tops. Over time, Tibetan anatomical knowledge found its way into Ayurveda [22] and to a lesser extent into Chinese medicine. [23] [24]

Christian Europe Edit

Throughout the history of Christian Europe, the dissection of human cadavers for medical education has experienced various cycles of legalization and proscription in different countries. Dissection was rare during the Middle Ages, but it was practised, [25] with evidence from at least as early as the 13th century. [26] [27] [28] The practice of autopsy in Medieval Western Europe is "very poorly known" as few surgical texts or conserved human dissections have survived. [29] A modern Jesuit scholar has claimed that the Christian theology contributed significantly to the revival of human dissection and autopsy by providing a new socio-religious and cultural context in which the human cadaver was no longer seen as sacrosanct. [26]

An edict of the 1163 Council of Tours, and an early 14th-century decree of Pope Boniface VIII have mistakenly been identified as prohibiting dissection and autopsy, misunderstanding or extrapolation from these edicts may have contributed to reluctance to perform such procedures. [30] [a] The Middle Ages witnessed the revival of an interest in medical studies, including human dissection and autopsy. [31]

Frederick II (1194–1250), the Holy Roman emperor, ruled that any that were studying to be a physician or a surgeon must attend a human dissection, which would be held no less than every five years. [8] Some European countries began legalizing the dissection of executed criminals for educational purposes in the late 13th and early 14th centuries. Mondino de Luzzi carried out the first recorded public dissection around 1315. [8] At this time, autopsies were carried out by a team consisting of a Lector, who lectured, the Sector, who did the dissection, and the Ostensor who pointed to features of interest. [8]

The Italian Galeazzo di Santa Sofia made the first public dissection north of the Alps in Vienna in 1404. [32]

Vesalius in the 16th century carried out numerous dissections in his extensive anatomical investigations. He was attacked frequently for his disagreement with Galen's opinions on human anatomy. Vesalius was the first to lecture and dissect the cadaver simultaneously. [8] [33]

The Catholic Church is known to have ordered an autopsy on conjoined twins Joana and Melchiora Ballestero in Hispaniola in 1533 to determine whether they shared a soul. They found that there were two distinct hearts, and hence two souls, based on the ancient Greek philosopher Empedocles, who believed the soul resided in the heart. [34]

Human dissection was also practised by Renaissance artists. Though most chose to focus on the external surfaces of the body, some like Michelangelo Buonarotti, Antonio del Pollaiolo, Baccio Bandinelli, and Leonardo da Vinci sought a deeper understanding. However, there were no provisions for artists to obtain cadavers, so they had to resort to unauthorised means, as indeed anatomists sometimes did, such as grave robbing, body snatching, and murder. [8]

Anatomization was sometimes ordered as a form of punishment, as, for example, in 1806 to James Halligan and Dominic Daley after their public hanging in Northampton, Massachusetts. [35]

In modern Europe, dissection is routinely practised in biological research and education, in medical schools, and to determine the cause of death in autopsy. It is generally considered a necessary part of learning and is thus accepted culturally. It sometimes attracts controversy, as when Odense Zoo decided to dissect lion cadavers in public before a "self-selected audience". [36] [37]

Britain Edit

In Britain, dissection remained entirely prohibited from the end of the Roman conquest and through the Middle Ages to the 16th century, when a series of royal edicts gave specific groups of physicians and surgeons some limited rights to dissect cadavers. The permission was quite limited: by the mid-18th century, the Royal College of Physicians and Company of Barber-Surgeons were the only two groups permitted to carry out dissections, and had an annual quota of ten cadavers between them. As a result of pressure from anatomists, especially in the rapidly growing medical schools, the Murder Act 1752 allowed the bodies of executed murderers to be dissected for anatomical research and education. By the 19th century this supply of cadavers proved insufficient, as the public medical schools were growing, and the private medical schools lacked legal access to cadavers. A thriving black market arose in cadavers and body parts, leading to the creation of the profession of body snatching, and the infamous Burke and Hare murders in 1828, when 16 people were murdered for their cadavers, to be sold to anatomists. The resulting public outcry led to the passage of the Anatomy Act 1832, which increased the legal supply of cadavers for dissection. [38]

By the 21st century, the availability of interactive computer programs and changing public sentiment led to renewed debate on the use of cadavers in medical education. The Peninsula College of Medicine and Dentistry in the UK, founded in 2000, became the first modern medical school to carry out its anatomy education without dissection. [39]

United States Edit

In the United States, dissection of frogs became common in college biology classes from the 1920s, and were gradually introduced at earlier stages of education. By 1988, some 75 to 80 percent of American high school biology students were participating in a frog dissection, with a trend towards introduction in elementary schools. The frogs are most commonly from the genus Rana. Other popular animals for high-school dissection at the time of that survey were, among vertebrates, fetal pigs, perch, and cats and among invertebrates, earthworms, grasshoppers, crayfish, and starfish. [40] About six million animals are (2016) dissected each year in United States high schools, not counting medical training and research. Most of these are purchased already dead from slaughterhouses and farms. [41]

Dissection in U.S. high schools became prominent in 1987, when a California student, Jenifer Graham, sued to require her school to let her complete an alternative project. The court ruled that mandatory dissections were permissible, but that Graham could ask to dissect a frog that had died of natural causes rather than one that was killed for the purposes of dissection the practical impossibility of procuring a frog that had died of natural causes in effect let Graham opt out of the required dissection. The suit gave publicity to anti-dissection advocates. Graham appeared in a 1987 Apple Computer commercial for the virtual-dissection software Operation Frog. [42] [43] The state of California passed a Student's Rights Bill in 1988 requiring that objecting students be allowed to complete alternative projects. [44] Opting out of dissection increased through the 1990s. [45]

In the United States, 17 states [b] along with Washington, D.C. have enacted dissection-choice laws or policies that allow students in primary and secondary education to opt out of dissection. Other states including Arizona, Hawaii, Minnesota, Texas, and Utah have more general policies on opting out on moral, religious, or ethical grounds. [46] To overcome these concerns, J. W. Mitchell High School in New Port Richey, Florida, in 2019 became the first US high school to use synthetic frogs for dissection in its science classes, instead of preserved real frogs. [47] [48] [49]

As for the dissection of cadavers in undergraduate and medical school, traditional dissection is supported by professors and students, with some opposition, limiting the availability of dissection. Upper-level students who have experienced this method along with their professors agree that "Studying human anatomy with colorful charts is one thing. Using a scalpel and an actual, recently-living person is an entirely different matter." [50]

The way in which cadaveric specimens are obtained differs greatly according to country. [51] In the UK, donation of a cadaver is wholly voluntary. Involuntary donation plays a role in about 20 percent of specimens in the US and almost all specimens donated in some countries such as South Africa and Zimbabwe. [51] Countries that practice involuntary donation may make available the bodies of dead criminals or unclaimed or unidentified bodies for the purposes of dissection. [51] Such practices may lead to a greater proportion of the poor, homeless and social outcasts being involuntarily donated. [51] Cadavers donated in one jurisdiction may also be used for the purposes of dissection in another, whether across states in the US, [4] or imported from other countries, such as with Libya. [51] As an example of how a cadaver is donated voluntarily, a funeral home in conjunction with a voluntary donation program identifies a body who is part of the program. After broaching the subject with relatives in a diplomatic fashion, the body is then transported to a registered facility. The body is tested for the presence of HIV and hepatitis viruses. It is then evaluated for use as a "fresh" or "prepared" specimen. [4]

Cadaveric specimens for dissection are, in general, disposed of by cremation. The deceased may then be interred at a local cemetery. If the family wishes, the ashes of the deceased are then returned to the family. [4] Many institutes have local policies to engage, support and celebrate the donors. This may include the setting up of local monuments at the cemetery. [4]

Human cadavers are often used in medicine to teach anatomy or surgical instruction. [4] [51] Cadavers are selected according to their anatomy and availability. They may be used as part of dissection courses involving a "fresh" specimen so as to be as realistic as possible—for example, when training surgeons. [4] Cadavers may also be pre-dissected by trained instructors. This form of dissection involves the preparation and preservation of specimens for a longer time period and is generally used for the teaching of anatomy. [4]

Some alternatives to dissection may present educational advantages over the use of animal cadavers, while eliminating perceived ethical issues. [52] These alternatives include computer programs, lectures, three dimensional models, films, and other forms of technology. Concern for animal welfare is often at the root of objections to animal dissection. [53] Studies show that some students reluctantly participate in animal dissection out of fear of real or perceived punishment or ostracism from their teachers and peers, and many do not speak up about their ethical objections. [54] [55]

One alternative to the use of cadavers is computer technology. At Stanford Medical School, software combines X-ray, ultrasound and MRI imaging for display on a screen as large as a body on a table. [56] In a variant of this, a "virtual anatomy" approach being developed at New York University, students wear three dimensional glasses and can use a pointing device to "[swoop] through the virtual body, its sections as brightly colored as living tissue." This method is claimed to be "as dynamic as Imax [cinema]". [57]

Proponents of animal-free teaching methodologies argue that alternatives to animal dissection can benefit educators by increasing teaching efficiency and lowering instruction costs while affording teachers an enhanced potential for the customization and repeat-ability of teaching exercises. Those in favor of dissection alternatives point to studies which have shown that computer-based teaching methods "saved academic and nonacademic staff time … were considered to be less expensive and an effective and enjoyable mode of student learning [and] … contributed to a significant reduction in animal use" because there is no set-up or clean-up time, no obligatory safety lessons, and no monitoring of misbehavior with animal cadavers, scissors, and scalpels. [58] [59] [60]

With software and other non-animal methods, there is also no expensive disposal of equipment or hazardous material removal. Some programs also allow educators to customize lessons and include built-in test and quiz modules that can track student performance. Furthermore, animals (whether dead or alive) can be used only once, while non-animal resources can be used for many years—an added benefit that could result in significant cost savings for teachers, school districts, and state educational systems. [58]

Several peer-reviewed comparative studies examining information retention and performance of students who dissected animals and those who used an alternative instruction method have concluded that the educational outcomes of students who are taught basic and advanced biomedical concepts and skills using non-animal methods are equivalent or superior to those of their peers who use animal-based laboratories such as animal dissection. [61] [62]

Some reports state that students’ confidence, satisfaction, and ability to retrieve and communicate information was much higher for those who participated in alternative activities compared to dissection. Three separate studies at universities across the United States found that students who modeled body systems out of clay were significantly better at identifying the constituent parts of human anatomy than their classmates who performed animal dissection. [63] [64] [65]

Another study found that students preferred using clay modeling over animal dissection and performed just as well as their cohorts who dissected animals. [66]

In 2008, the National Association of Biology Teachers (NABT) affirmed its support for classroom animal dissection stating that they "Encourage the presence of live animals in the classroom with appropriate consideration to the age and maturity level of the students …NABT urges teachers to be aware that alternatives to dissection have their limitations. NABT supports the use of these materials as adjuncts to the educational process but not as exclusive replacements for the use of actual organisms." [67]

The National Science Teachers Association (NSTA) "supports including live animals as part of instruction in the K-12 science classroom because observing and working with animals firsthand can spark students' interest in science as well as a general respect for life while reinforcing key concepts" of biological sciences. NSTA also supports offering dissection alternatives to students who object to the practice. [68]

The NORINA database lists over 3,000 products which may be used as alternatives or supplements to animal use in education and training. [69] These include alternatives to dissection in schools. InterNICHE has a similar database and a loans system. [70]


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Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. / Collins, Sean R. Miller, Kyle M. Maas, Nancy L. Roguev, Assen Fillingham, Jeffrey Chu, Clement S. Schuldiner, Maya Gebbia, Marinella Recht, Judith Shales, Michael Ding, Huiming Xu, Hong Han, Junhong Ingvarsdottir, Kristin Cheng, Benjamin Andrews, Brenda Boone, Charles Berger, Shelley L. Hieter, Phil Zhang, Zhiguo Brown, Grant W. Ingles, C. James Emili, Andrew Allis, C. David Toczyski, David P. Weissman, Jonathan S. Greenblatt, Jack F. Krogan, Nevan J.

In: Nature , Vol. 446, No. 7137, 12.04.2007, p. 806-810.

Research output : Contribution to journal › Article › peer-review

T1 - Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map

AU - Ingvarsdottir, Kristin

N2 - Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

AB - Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.


Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map

Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.


What is functional dissection? - Biology

a Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK
E-mail: [email protected]

b Institute for Molecular Precision Medicine, Xiangya Hospital, Central South University, Changsha, China

Abstract

Protein O-GlcNAcylation is an abundant post-translational modification of intracellular proteins with the monosaccharide N-acetylglucosamine covalently tethered to serines and threonines. Modification of proteins with O-GlcNAc is required for metazoan embryo development and maintains cellular homeostasis through effects on transcription, signalling and stress response. While disruption of O-GlcNAc homeostasis can have detrimental impact on cell physiology and cause various diseases, little is known about the functions of individual O-GlcNAc sites. Most of the sites are modified sub-stoichiometrically which is a major challenge to the dissection of O-GlcNAc function. Here, we discuss the application, advantages and limitations of the currently available tools and technologies utilised to dissect the function of O-GlcNAc on individual proteins and sites in vitro and in vivo. Additionally, we provide a perspective on future developments required to decipher the protein- and site-specific roles of this essential sugar modification.


Identification and Functional Dissection of Stress-responsive Genes in Cotton

Cotton is one of the most important fiber and oil crops. Abiotic stress (salt stress, drought stress, heat stress and chilling stress) and biotic stress (Verticillium wilt, fusarium wilt, pests) severely threaten cotton productivity. Most commercial cotton cultivars show poor resistance to Verticillium wilt, .

Cotton is one of the most important fiber and oil crops. Abiotic stress (salt stress, drought stress, heat stress and chilling stress) and biotic stress (Verticillium wilt, fusarium wilt, pests) severely threaten cotton productivity. Most commercial cotton cultivars show poor resistance to Verticillium wilt, resulting in decreased fiber quality and annual crop yield, with losses reaching 30%-80% in severe disease outbreaks. Additionally, global climate change, drought, heat, and chilling stress dramatically suppress cotton growth and productivity. To develop germplasm that are abiotic and biotic stress resistant remains enigmatic. For sustained cotton breeding, it is essential to develop elite cotton varieties with enhanced tolerance to biotic and abiotic stress, without reduction in quality.

The location of elite alleles and loci with increased tolerance to biotic and abiotic stress will accelerate cotton breeding. It is also possible to study such elite genes originating from wild cotton, sea island cotton, other stress-tolerant plants, or microorganisms to enhance cotton stress tolerance. The function of these candidate genes or loci can be verified by genetic and biochemical processes. Genetic and biochemical analyses confirm the function of these candidate genes or loci, which are aggregated to produce elite cotton germplasm material.

The goal of this Research Topic is to summarize the advances in elite gene screening and the production of germplasm material for cotton tolerance to abiotic and biotic stress.

Specific themes include, but not limited to:
• Summary of the progress about cotton abiotic and biotic stress study, present the main challenges faced by cotton stress tolerant breeding, as well as the solutions, and future breeding strategies (reviews on invitation)
• Identification and functional characterization of elite alleles controlling abiotic stress including drought, salt stress, and elite germplasm materials creation with improved stress tolerance but without production penalty
• Identification and functional characterization of effector from Verticillium wilt, fusarium wilt and interacted proteins in plants, creation disease-resistant materials using transgenic technology and gene editing
• Identification and functional characterization of elite alleles and loci related to abiotic and biotic stress through population, omics, and other analysis,

We specifically welcome field-based studies studying the effects of cotton stress in the field as supposed to the lab.

Studies falling into the categories below will not be considered for review, unless they are expanded and provide insight into the biological system or process being studied:

i) Descriptive collection of transcripts, proteins or metabolites, including comparative sets as a result of different conditions or treatments
ii) Descriptive studies that define gene families using basic phylogenetics and the assignment of cursory functional attributions (e.g. expression profiles, hormone or metabolites levels, promoter analysis, informatic parameters)
ii) Descriptive studies using -omics approaches

The Topic Editors would like to acknowledge Dr. Xiaoyang Ge from State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS for his contribution in the organization of this Research Topic.

Keywords: Abiotic and Biotic Stress, Cotton, Verticillium wilt, Germplasm creation, Resistance

Important Note: All contributions to this Research Topic must be within the scope of the section and journal to which they are submitted, as defined in their mission statements. Frontiers reserves the right to guide an out-of-scope manuscript to a more suitable section or journal at any stage of peer review.


Acknowledgements

We are grateful to K. Tipton and M. Bassik for critically reading the manuscript, S. Gasser, B. Frey and Vincent Cheung for discussion, and G. Narlikar for reagents. We thank N. Datta, T. Punna, N. Thompson, M. Ballantine, N. Gabovic, A. Wind, K. Chin, Y. Xue, A. Chan, Y. Xue, T. Chan, M. Xan, M. Lim, H. Dalgleish, K. Vachon, L. Le, C. Sun, Z. Hassam, J. Rilestone and K. Takhar for technical assistance. We also thank S. Jackson, Z. Zhang, Vanessa Cheung, F. Winston, J. Erkmann and P. Kaufman for communicating results before publication. This research was supported by grants from Genome Canada and the Ontario Genomics Institute (J.F.G., A.E., C.B. and B.A.), the NIH (D.P.T.), the Howard Hughes Medical Institute (J.S.W.) and the Canadian Institute of Health Research (N.J.K., C.J.I. and G.W.B.). S.R.C. was funded by a fellowship from the Burroughs Wellcome Fund. N.J.K. is a Sandler Family Fellow.


SPECIFIC TARGETING OF GLUTAMATERGIC AND GABAERGIC BNST SUBPOPULATIONS

Glutamate and gamma-aminobutyric acid (GABA) are the principal excitatory and inhibitory neurotransmitters in the brain, respectively. The anterior and dorsal parts of the BNST are mostly comprised of GABAergic neurons, while the posterior and ventral parts of the BNST contain significant numbers of both glutamatergic and GABAergic neurons (Poulin et al., 2009). To target glutamatergic or GABAergic BNST subpopulations, most studies used the Vglut2 or Vgat gene (that encodes vesicular glutamate transporter 2 or vesicular GABA transporter, respectively) as the genetic marker, respectively (Bhatti et al., 2020 Jennings et al., 2013a 2013b), while some used Gad2 (encoding 65 kDa isoform of glutamic acid decarboxylase) to gain genetic access to GABAergic BNST neurons (Hao et al., 2019). Specifically, these studies used knock-in mouse lines expressing a bacterial recombinase Cre under the promoter of Vglut2, Vgat, or Gad2 in combination with viral tools that allow Cre-dependent expression of optogenetic or chemogenetic tools or fluorescent calcium indicators. Notably, all the studies have demonstrated that optogenetic stimulation of each neural population indeed evoked the anticipated excitatory or inhibitory responses in the postsynaptic neurons, confirming the tight correspondence between molecular and electrophysiological phenotypes.


Functional dissection of the NuA4 histone acetyltransferase reveals its role as a genetic hub and that Eaf1 is essential for complex integrity

The Saccharomyces cerevisiae NuA4 histone acetyltransferase complex catalyzes the acetylation of histone H4 and the histone variant Htz1 to regulate key cellular events, including transcription, DNA repair, and faithful chromosome segregation. To further investigate the cellular processes impacted by NuA4, we exploited the nonessential subunits of the complex to build an extensive NuA4 genetic-interaction network map. The map reveals that NuA4 is a genetic hub whose function buffers a diverse range of cellular processes, many not previously linked to the complex, including Golgi complex-to-vacuole vesicle-mediated transport. Further, we probe the role that nonessential subunits play in NuA4 complex integrity. We find that most nonessential subunits have little impact on NuA4 complex integrity and display between 12 and 42 genetic interactions. In contrast, the deletion of EAF1 causes the collapse of the NuA4 complex and displays 148 genetic interactions. Our study indicates that Eaf1 plays a crucial function in NuA4 complex integrity. Further, we determine that Eaf5 and Eaf7 form a subcomplex, which reflects their similar genetic interaction profiles and phenotypes. Our integrative study demonstrates that genetic interaction maps are valuable in dissecting complex structure and provides insight into why the human NuA4 complex, Tip60, has been associated with a diverse range of pathologies.

Figures

Synthetic genetic-interaction map of five…

Synthetic genetic-interaction map of five NuA4 subunits. Genome-wide SL-SGA screens were performed using…

Eaf1-TAP purifies the NuA4 complex.…

Eaf1-TAP purifies the NuA4 complex. SDS-PAGE (gradient gel) and silver staining comparing NuA4…

NuA4 function impacts vesicle-mediated transport.…

NuA4 function impacts vesicle-mediated transport. (A) eaf1 Δ, yaf9 Δ, yng2 Δ, and…

NuA4 physically interacts with Msn4…

NuA4 physically interacts with Msn4 but does not regulate Msn4 binding to the…

Eaf1 is required for NuA4…

Eaf1 is required for NuA4 complex integrity. SDS-PAGE (gradient gel) and silver staining…

Eaf5 and Eaf7 form a subcomplex within NuA4. (A) Two-dimensional, hierarchical clustering of…


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Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. / Collins, Sean R. Miller, Kyle M. Maas, Nancy L. Roguev, Assen Fillingham, Jeffrey Chu, Clement S. Schuldiner, Maya Gebbia, Marinella Recht, Judith Shales, Michael Ding, Huiming Xu, Hong Han, Junhong Ingvarsdottir, Kristin Cheng, Benjamin Andrews, Brenda Boone, Charles Berger, Shelley L. Hieter, Phil Zhang, Zhiguo Brown, Grant W. Ingles, C. James Emili, Andrew Allis, C. David Toczyski, David P. Weissman, Jonathan S. Greenblatt, Jack F. Krogan, Nevan J.

In: Nature , Vol. 446, No. 7137, 12.04.2007, p. 806-810.

Research output : Contribution to journal › Article › peer-review

T1 - Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map

AU - Ingvarsdottir, Kristin

N1 - Funding Information: Acknowledgements We are grateful to K. Tipton and M. Bassik for critically reading the manuscript, S. Gasser, B. Frey and Vincent Cheung for discussion, and G. Narlikar for reagents. We thank N. Datta, T. Punna, N. Thompson, M. Ballantine, N. Gabovic, A. Wind, K. Chin, Y. Xue, A. Chan, Y. Xue, T. Chan, M. Xan, M. Lim, H. Dalgleish, K. Vachon, L. Le, C. Sun, Z. Hassam, J. Rilestone and K. Takhar for technical assistance. We also thank S. Jackson, Z. Zhang, Vanessa Cheung, F. Winston, J. Erkmann and P. Kaufman for communicating results before publication. This research was supported by grants from Genome Canada and the Ontario Genomics Institute (J.F.G., A.E., C.B. and B.A.), the NIH (D.P.T.), the Howard Hughes Medical Institute (J.S.W.) and the Canadian Institute of Health Research (N.J.K., C.J.I. and G.W.B.). S.R.C. was funded by a fellowship from the Burroughs Wellcome Fund. N.J.K. is a Sandler Family Fellow. Copyright: Copyright 2015 Elsevier B.V., All rights reserved.

N2 - Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.

AB - Defining the functional relationships between proteins is critical for understanding virtually all aspects of cell biology. Large-scale identification of protein complexes has provided one important step towards this goal however, even knowledge of the stoichiometry, affinity and lifetime of every protein-protein interaction would not reveal the functional relationships between and within such complexes. Genetic interactions can provide functional information that is largely invisible to protein-protein interaction data sets. Here we present an epistatic miniarray profile (E-MAP) consisting of quantitative pairwise measurements of the genetic interactions between 743 Saccharomyces cerevisiae genes involved in various aspects of chromosome biology (including DNA replication/repair, chromatid segregation and transcriptional regulation). This E-MAP reveals that physical interactions fall into two well-represented classes distinguished by whether or not the individual proteins act coherently to carry out a common function. Thus, genetic interaction data make it possible to dissect functionally multi-protein complexes, including Mediator, and to organize distinct protein complexes into pathways. In one pathway defined here, we show that Rtt109 is the founding member of a novel class of histone acetyltransferases responsible for Asf1-dependent acetylation of histone H3 on lysine 56. This modification, in turn, enables a ubiquitin ligase complex containing the cullin Rtt101 to ensure genomic integrity during DNA replication.