WorldWideScience

Sample records for human cloning prohibition

  1. Human embryo cloning prohibited in Hong Kong.

    Science.gov (United States)

    Liu, Athena

    2005-12-01

    Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

  2. Cloning Mice and Men: Prohibiting the Use of iPS Cells for Human Reproductive Cloning

    OpenAIRE

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2010-01-01

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

  3. Cloning mice and men: prohibiting the use of iPS cells for human reproductive cloning.

    Science.gov (United States)

    Lo, Bernard; Parham, Lindsay; Alvarez-Buylla, Arturo; Cedars, Marcelle; Conklin, Bruce; Fisher, Susan; Gates, Elena; Giudice, Linda; Halme, Dina Gould; Hershon, William; Kriegstein, Arnold; Kwok, Pui-Yan; Wagner, Richard

    2010-01-08

    The use of iPSCs and tetraploid complementation for human reproductive cloning would raise profound ethical objections. Professional standards and laws that ban human reproductive cloning by somatic cell nuclear transfer should be revised to also forbid it by other methods, such as iPSCs via tetraploid complementation.

  4. Islamic perspectives on human cloning.

    Science.gov (United States)

    Sadeghi, Mahmoud

    2007-01-01

    The present paper seeks to assess various views from Islamic jurists relating to human cloning, which is one of the controversial topics in the recent past. Taking Islamic jurisprudence principles, such as the rule of necessity for self preservation and respect for human beings, the rule of la darar wa la dirar ('the necessity to refrain from causing harm to oneself and others') and the rule of usr wa haraj, one may indicate that if human cloning could not be prohibited, as such, it could still be opposed because it gives way to various harmful consequences, which include family disorder, chaos in the clone's family relationships, physical and mental diseases for clones and suffering of egg donors and surrogate mothers. However with due attention to the fact that the reasons behind the prohibition of abortion only restrict the destruction of human embryos in their post-implantation stages, human cloning for biomedical research and exploitation of stem cells from cloned embryos at the blastocyst stage for therapeutic purposes would be acceptable.

  5. Human Cloning

    Science.gov (United States)

    2006-07-20

    genes , for example, has led to new treatments developed by the biotechnology industry for diseases such as diabetes and hemophilia . In the context of...stem cells should be permitted because of the potential for developing new therapies and advancing biomedical knowledge. On May 24, 2005, the House...to describe many different processes that involve making copies of biological material, such as a gene , a cell, a plant or an animal. The cloning of

  6. Statement on Human Cloning

    Science.gov (United States)

    ... ban on efforts to implant a human cloned embryo for the purpose of reproduction. The scientific evidence ... stem cell research, including the use of nuclear transplantation techniques (also known as research or therapeutic cloning), ...

  7. BIOETHICS AND HUMAN CLONING

    Directory of Open Access Journals (Sweden)

    Željko Kaluđerović

    2011-12-01

    Full Text Available In this paper the authors analyze the process of negotiating and beginning of the United Nations Declaration on Human Cloning as well as the paragraphs of the very Declaration. The negotiation was originally conceived as a clear bioethical debate that should have led to a general agreement to ban human cloning. However, more often it had been discussed about human rights, cultural, civil and religious differences between people and about priorities in case of eventual conflicts between different value systems. In the end, a non-binding Declaration on Human Cloning had been adopted, full of numerous compromises and ambiguous formulations, that relativized the original intention of proposer states. According to authors, it would have been better if bioethical discussion and eventual regulations on cloning mentioned in the following text had been left over to certain professional bodies, and only after the public had been fully informed about it should relevant supranational organizations have taken that into consideration.

  8. The First Human Cloned Embryo.

    Science.gov (United States)

    Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

    2002-01-01

    Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

  9. [Scientific ethics of human cloning].

    Science.gov (United States)

    Valenzuela, Carlos Y

    2005-01-01

    True cloning is fission, budding or other types of asexual reproduction. In humans it occurs in monozygote twinning. This type of cloning is ethically and religiously good. Human cloning can be performed by twinning (TWClo) or nuclear transfer (NTClo). Both methods need a zygote or a nuclear transferred cell, obtained in vitro (IVTec). They are under the IVTec ethics. IVTecs use humans (zygotes, embryos) as drugs or things; increase the risk of malformations; increase development and size of abnormalities and may cause long-term changes. Cloning for preserving extinct (or almost extinct) animals or humans when sexual reproduction is not possible is ethically valid. The previous selection of a phenotype in human cloning violates some ethical principles. NTClo for reproductive or therapeutic purposes is dangerous since it increases the risk for nucleotide or chromosome mutations, de-programming or re-programming errors, aging or malignancy of the embryo cells thus obtained.

  10. Statement on Human Cloning

    Science.gov (United States)

    ... as our understanding of this technology advances. Support Stem Cell Research (including Research Cloning) AAAS supports stem cell research, including the use of nuclear transplantation techniques (also ...

  11. Human cloning and child welfare.

    Science.gov (United States)

    Burley, J; Harris, J

    1999-01-01

    In this paper we discuss an objection to human cloning which appeals to the welfare of the child. This objection varies according to the sort of harm it is expected the clone will suffer. The three formulations of it that we will consider are: 1. Clones will be harmed by the fearful or prejudicial attitudes people may have about or towards them (H1); 2. Clones will be harmed by the demands and expectations of parents or genotype donors (H2); 3. Clones will be harmed by their own awareness of their origins, for example the knowledge that the genetic donor is a stranger (H3). We will show why these three versions of the child welfare objection do not necessarily supply compelling reasons to ban human reproductive cloning. The claim that we will develop and defend in the course of our discussion is that even if it is the case that a cloned child will suffer harms of the type H1-H3, it is none the less permissible to conceive by cloning so long as these cloning-induced welfare deficits are not such as to blight the existence of the resultant child, whoever this may be. PMID:10226914

  12. Human cloning and 'posthuman' society.

    Science.gov (United States)

    Blackford, Russell

    2005-01-01

    Since early 1997, when the creation of Dolly the sheep by somatic cell nuclear transfer was announced in Nature, numerous government reports, essays, articles and books have considered the ethical problems and policy issues surrounding human reproductive cloning. In this article, I consider what response a modern liberal society should give to the prospect of human cloning, if it became safe and practical. Some opponents of human cloning have argued that permitting it would place us on a slippery slope to a repugnant future society, comparable to that portrayed in Aldous Huxley's novel, Brave New World. I conclude that, leaving aside concerns about safety, none of the psychological or social considerations discussed in this article provides an adequate policy justification for invoking the state's coercive powers to prevent human cloning.

  13. Human cloning, stem cell research. An Islamic perspective.

    Science.gov (United States)

    Al-Aqeel, Aida I

    2009-12-01

    The rapidly changing technologies that involve human subjects raise complex ethical, legal, social, and religious issues. Recent advances in the field of cloning and stem cell research have introduced new hopes for the treatment of serious diseases. But this promise has raised many complex questions. This field causes debate and challenge, not only among scientists but also among ethicists, religious scholars, governments, and politicians. There is no consensus on the morality of human cloning, even within specific religious traditions. In countries in which religion has a strong influence on political decision making, the moral status of the human embryo is at the center of the debate. Because of the inevitable consequences of reproductive cloning, it is prohibited in Islam. However, stem cell research for therapeutic purposes is permissible with full consideration, and all possible precautions in the pre-ensoulment stages of early fetus development, if the source is legitimate.

  14. The global governance of human cloning: the case of UNESCO

    Science.gov (United States)

    Langlois, Adèle

    2017-01-01

    Since Dolly the Sheep was cloned in 1996, the question of whether human reproductive cloning should be banned or pursued has been the subject of international debate. Feelings run strong on both sides. In 2005, the United Nations adopted its Declaration on Human Cloning to try to deal with the issue. The declaration is ambiguously worded, prohibiting “all forms of human cloning inasmuch as they are incompatible with human dignity and the protection of human life”. It received only ambivalent support from UN member states. Given this unsatisfactory outcome, in 2008 UNESCO (the United Nations Educational, Scientific and Cultural Organization) set up a Working Group to investigate the possibility of a legally binding convention to ban human reproductive cloning. The Working Group was made up of members of the International Bioethics Committee, established in 1993 as part of UNESCO’s Bioethics Programme. It found that the lack of clarity in international law is unhelpful for those states yet to formulate national regulations or policies on human cloning. Despite this, member states of UNESCO resisted the idea of a convention for several years. This changed in 2015, but there has been no practical progress on the issue. Drawing on official records and first-hand observations at bioethics meetings, this article examines the human cloning debate at UNESCO from 2008 onwards, thus building on and advancing current scholarship by applying recent ideas on global governance to an empirical case. It concludes that, although human reproductive cloning is a challenging subject, establishing a robust global governance framework in this area may be possible via an alternative deliberative format, based on knowledge sharing and feasibility testing rather than the interest-based bargaining that is common to intergovernmental organizations and involving a wide range of stakeholders. This article is published as part of a collection on global governance.

  15. The global governance of human cloning: the case of UNESCO.

    Science.gov (United States)

    Langlois, Adèle

    2017-03-21

    Since Dolly the Sheep was cloned in 1996, the question of whether human reproductive cloning should be banned or pursued has been the subject of international debate. Feelings run strong on both sides. In 2005, the United Nations adopted its Declaration on Human Cloning to try to deal with the issue. The declaration is ambiguously worded, prohibiting "all forms of human cloning inasmuch as they are incompatible with human dignity and the protection of human life". It received only ambivalent support from UN member states. Given this unsatisfactory outcome, in 2008 UNESCO (the United Nations Educational, Scientific and Cultural Organization) set up a Working Group to investigate the possibility of a legally binding convention to ban human reproductive cloning. The Working Group was made up of members of the International Bioethics Committee, established in 1993 as part of UNESCO's Bioethics Programme. It found that the lack of clarity in international law is unhelpful for those states yet to formulate national regulations or policies on human cloning. Despite this, member states of UNESCO resisted the idea of a convention for several years. This changed in 2015, but there has been no practical progress on the issue. Drawing on official records and first-hand observations at bioethics meetings, this article examines the human cloning debate at UNESCO from 2008 onwards, thus building on and advancing current scholarship by applying recent ideas on global governance to an empirical case. It concludes that, although human reproductive cloning is a challenging subject, establishing a robust global governance framework in this area may be possible via an alternative deliberative format, based on knowledge sharing and feasibility testing rather than the interest-based bargaining that is common to intergovernmental organizations and involving a wide range of stakeholders. This article is published as part of a collection on global governance.

  16. Cloning humans? Current science, current views, and a perspective from Christianity.

    Science.gov (United States)

    Brun, Rudolf B

    2002-01-01

    Therapeutic cloning is urgent and should be vigorously supported. To successfully argue for this position, the distinction between a human embryo and a human nuclear transplant may be helpful. Even if current technical difficulties should be solved, global legislation should prohibit cloning for the purpose of fabricating babies. This position originates from a view on human nature in general and from a Christian perspective in particular.

  17. Human reproductive cloning: a conflict of liberties.

    Science.gov (United States)

    Havstad, Joyce C

    2010-02-01

    Proponents of human reproductive cloning do not dispute that cloning may lead to violations of clones' right to self-determination, or that these violations could cause psychological harms. But they proceed with their endorsement of human reproductive cloning by dismissing these psychological harms, mainly in two ways. The first tactic is to point out that to commit the genetic fallacy is indeed a mistake; the second is to invoke Parfit's non-identity problem. The argument of this paper is that neither approach succeeds in removing our moral responsibility to consider and to prevent psychological harms to cloned individuals. In fact, the same commitment to personal liberty that generates the right to reproduce by means of cloning also creates the need to limit that right appropriately. Discussion of human reproductive cloning ought to involve a careful and balanced consideration of both the relevant aspects of personal liberty - the parents' right to reproductive freedom and the cloned child's right to self-determination.

  18. "Goodbye Dolly?" The ethics of human cloning.

    Science.gov (United States)

    Harris, J

    1997-01-01

    The ethical implications of human clones have been much alluded to, but have seldom been examined with any rigour. This paper examines the possible uses and abuses of human cloning and draws out the principal ethical dimensions, both of what might be done and its meaning. The paper examines some of the major public and official responses to cloning by authorities such as President Clinton, the World Health Organisation, the European parliament, UNESCO, and others and reveals their inadequacies as foundations for a coherent public policy on human cloning. The paper ends by defending a conception of reproductive rights of "procreative autonomy" which shows human cloning to be not inconsistent with human rights and dignity. PMID:9451604

  19. What exactly is an exact copy? And why it matters when trying to ban human reproductive cloning in Australia.

    Science.gov (United States)

    Gogarty, B

    2003-04-01

    This paper examines the current Australian regulatory response to human reproductive cloning. The central consideration is the capacity of the current regulatory regime to effectively deter human cloning efforts. A legislative prohibition on human cloning must be both effective and clear enough to allow researchers to know what practices are acceptable. This paper asks whether the current Australian regime evinces these qualities and suggests that Australia should follow the example set in the UK by the enactment of the Human Reproductive Cloning Act 2001.

  20. The ethics of human reproductive cloning.

    Science.gov (United States)

    Strong, Carson

    2005-03-01

    This article addresses the question of whether human reproductive cloning could be ethically justifiable in at least some cases involving infertile couples who would choose cloning as a way to have a genetically related child. At present, the risk of congenital anomalies constitutes a compelling argument against human reproductive cloning. The article explores whether reproductive cloning could be ethically justifiable if, at some future time, cloning becomes possible without an elevated risk of anomalies. It is argued that freedom to use cloning is a form of procreative freedom and, as such, deserves respect. All of the objections that have been raised against human reproductive cloning fall under three main categories: those that appeal to the interests of the child, those based on consequences for society, and those arising from teleological views. Objections that appeal to the child's interests are, in turn, of two main kinds: consequentialist and deontological. All of these types of objections are examined, and it is found that each involves serious problems that prevent it from being a reasonable objection in the context of the infertility cases considered. It is concluded that human reproductive cloning would be ethically justifiable in at least some cases involving infertile couples, provided that it could be performed without an elevated risk of anomalies.

  1. Human Cloning: Let's Discuss It.

    Science.gov (United States)

    Taras, Loretta; Stavroulakis, Anthea M.; Ortiz, Mary T.

    1999-01-01

    Describes experiences with holding discussions on cloning at a variety of levels in undergraduate biology courses. Discusses teaching methods used and student reactions to the discussions. Contains 12 references. (WRM)

  2. [Human cloning in Muslim and Arab law].

    Science.gov (United States)

    Aldeeb Abu-Sahlieh, Sami A

    2009-01-01

    Cloning is a modern medical procedure that Muslim religious authorities treat en resorting to the general principles established by classical Muslim law based on the Koran and the Sunnah of Muhhamad as the messenger of God. In this regard, human beings are not capable of deciding what is or what is not lawful without resorting to divine norms. Cloning clashes with several principles. Firstly, the principle of the respect for life in relation to surpernumeraries, but Muslim authors are not in unanimous agreement on the determination of the moment at which life begins. Secondly, is the respect of progeny: cloning could only take place between a married couple. But even if these two principles are respected, cloning poses two major problems: the diversity of species expounded by the Koran and the Sunnah and a lack of interest. Which explains the quasi-unanimous opposition of Muslim writings regarding cloning.

  3. Emotional reactions to human reproductive cloning.

    Science.gov (United States)

    May, Joshua

    2016-01-01

    Extant surveys of people's attitudes towards human reproductive cloning focus on moral judgements alone, not emotional reactions or sentiments. This is especially important given that some (especially Leon Kass) have argued against such cloning on the ground that it engenders widespread negative emotions, like disgust, that provide a moral guide. To provide some data on emotional reactions to human cloning, with a focus on repugnance, given its prominence in the literature. This brief mixed-method study measures the self-reported attitudes and emotions (positive or negative) towards cloning from a sample of participants in the USA. Most participants condemned cloning as immoral and said it should be illegal. The most commonly reported positive sentiment was by far interest/curiosity. Negative emotions were much more varied, but anxiety was the most common. Only about a third of participants selected disgust or repugnance as something they felt, and an even smaller portion had this emotion come to mind prior to seeing a list of options. Participants felt primarily interested and anxious about human reproductive cloning. They did not primarily feel disgust or repugnance. This provides initial empirical evidence that such a reaction is not appropriately widespread. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  4. Cloning

    Science.gov (United States)

    ... copies of whole animals Therapeutic cloning, which creates embryonic stem cells. Researchers hope to use these cells to grow healthy tissue to replace injured or diseased tissues in the human body. NIH: National Human Genome Research Institute

  5. Human reproductive cloning and reasons for deprivation.

    Science.gov (United States)

    Jensen, D A

    2008-08-01

    Human reproductive cloning provides the possibility of genetically related children for persons for whom present technologies are ineffective. I argue that the desire for genetically related children is not, by itself, a sufficient reason to engage in human reproductive cloning. I show this by arguing that the value underlying the desire for genetically related children implies a tension between the parent and the future child. This tension stems from an instance of a deprivation and violates a general principle of reasons for deprivation. Alternative considerations, such as a right to procreative autonomy, do not appear helpful in making the case for human reproductive cloning merely on the basis of the desire for genetically related children.

  6. MOLECULAR CLONING OF HUMAN NEUROTROPHIN-4 GENE

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Cloning and sequencing of the human neurotrophin-4(hNT-4) gene.Methods With the chromosomal DNA of human blood lymphocytes as template,hNT-4 coding genes were amplified by polymerase chain reaction(PCR) and recombinated into phage vector pGEM-T Easy,which were sequenced by using Sanger's single stranded DNA terminal termination method.Results The sequence of the cloned gene is completely the same as that reported in the literature(GenBank data base,M86528).Conclusion This study successfully cloning and sequenced the gene of mhNT-4,and it would be convenient for us to study the expression of mhNT-4 in eukaryote,and to continue the research on the gene therapy of Alzheimer's disease intensively.This study indicate that the hNT-4 is conservative in different races and individuals.

  7. Human cloning: three mistakes and an alternative.

    Science.gov (United States)

    Baylis, Françoise

    2002-06-01

    The current debate on the ethics of cloning humans is both uninspired and uninspiring. In large measure this is because of mistakes that permeate the discourse, including the mistake of thinking that cloning technology is strictly a reproductive technology when it is used to create whole beings. As a result, the challenge this technology represents regarding our understanding of ourselves and the species to which we belong typically is inappropriately downplayed or exaggerated. This has meant that important (albeit disquieting) societal issues and species-type concerns have not been fully explored. This paper, intended as a corrective, suggests that we take an alternate view of human cloning as both an enhancement and a reproductive technology. This proposed shift in the framework for analysis counters the current narrow framing of the issues and introduces new questions about the prospect of modifying the species.

  8. [Ethical considerations on human cloning. A psychoanalytic perspective].

    Science.gov (United States)

    Alvarez, A

    2000-01-01

    A brief review of ethical issues related to two types of human cloning is presented: cloning embryonic cells not intended to culminate in the birth of a new individual and cloning human beings. Advantages and objections related to both types of human cloning are analyzed from an ethical point of view. Repercussions on individuals born by the technique of cloning are discussed from a psychoanalytical perspective. It can be concluded that cloning embryonic cells could be admissible, while not cloning considered as a reproductive option.

  9. THE CLONING OF HUMAN NEUROTROPHIN-3 GENE

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    In the present study, we have cloned the gene of human neurotrophin-3 (hNT-3) from the genomic DNA of white blood cells (WBC) by polymerase chain reaction (PCR). The amplification products were cloned into pUC19 and sequenced. Genomic sequence comparison of the cloned fragment and the reported hNT-3 (GenBank M61180) reveals 7 base differences: 1 in the signal peptide, 3 in the prepro peptide, and 3 in the mature hNT-3. Except the 2 varied bases (16th, T to G; 285th, A to C) in the signal peptide and pro-sequence resulted in the change of their encoded amino-acids (Tyr→Asp; Gln→His), the other varied bases have no influence on their respective encoded amino-acids, and all the changes have no influence on the open reading frame (ORF) of the hNT-3.

  10. Cloning for human reproduction: one American perspective.

    Science.gov (United States)

    Chester, R

    2001-09-01

    The author, an American law professor, believes that whole-body cloning of adult humans will be possible in the near future. He does not believe the procedure should be banned when used as a form of assisted reproduction, but that it should be regulated by the government to ensure proper testing and application. After raising a number of scientific, ethical, religious and legal issues, Professor Chester addresses parentage in light of both old and new concepts of the 'family.' Finally, he focuses on the problem of women as surrogate mothers of clones, arguing in the process that the surrogate, having no real genetic tie to the clone, would have less of a claim to parentage than at least some of the surrogates currently gestating foetuses.

  11. Cloning humans? Biological, ethical, and social considerations.

    Science.gov (United States)

    Ayala, Francisco J

    2015-07-21

    There are, in mankind, two kinds of heredity: biological and cultural. Cultural inheritance makes possible for humans what no other organism can accomplish: the cumulative transmission of experience from generation to generation. In turn, cultural inheritance leads to cultural evolution, the prevailing mode of human adaptation. For the last few millennia, humans have been adapting the environments to their genes more often than their genes to the environments. Nevertheless, natural selection persists in modern humans, both as differential mortality and as differential fertility, although its intensity may decrease in the future. More than 2,000 human diseases and abnormalities have a genetic causation. Health care and the increasing feasibility of genetic therapy will, although slowly, augment the future incidence of hereditary ailments. Germ-line gene therapy could halt this increase, but at present, it is not technically feasible. The proposal to enhance the human genetic endowment by genetic cloning of eminent individuals is not warranted. Genomes can be cloned; individuals cannot. In the future, therapeutic cloning will bring enhanced possibilities for organ transplantation, nerve cells and tissue healing, and other health benefits.

  12. Cell phoney: human cloning after Quintavalle.

    Science.gov (United States)

    Morgan, Derek; Ford, Mary

    2004-12-01

    Reproductive cloning has thrown up new scientific possibilities, ethical conundrums, and legal challenges. An initial question, considered by the English courts in 2003, was whether the technique presently available, that of cell nucleus replacement, falls outside the provisions of the Human Fertilisation and Embryology Act 1990. If it does, the creation and use, including use in research protocols, of human embryos would be unregulated, disclosing a need to consider remedial legislation. The resolution by the courts of this legal question dramatically engages them in a resolution of fundamental ethical dilemmas, and discloses the possibilities and limitation of negotiating science policy through the processes of litigation.

  13. Developing a code of ethics for human cloning.

    Science.gov (United States)

    Collmann, J; Graber, G

    2000-01-01

    Under what conditions might the cloning of human beings constitute an ethical practice? A tendency exists to analyze human cloning merely as a technical procedure. As with all revolutionary technological developments, however, human cloning potentially exists in a broad social context that will both shape and be shaped by the biological techniques. Although human cloning must be subjected to technical analysis that addresses fundamental ethical questions such as its safety and efficacy, questions exist that focus our attention on broader issues. Asserting that cloning inevitably leads to undesirable consequences commits the fallacy of technological determinism and untenably separates technological and ethical evaluation. Drawing from the Report of the National Bioethics Advisory Committee and Aldous Huxley's Brave New World, we offer a draft "Code of Ethics for Human Cloning" in order to stimulate discussion about the ethics of the broader ramifications of human cloning as well as its particular technological properties.

  14. Ethical issues regarding human cloning: a nursing perspective.

    Science.gov (United States)

    Dinç, Leyla

    2003-05-01

    Advances in cloning technology and successful cloning experiments in animals raised concerns about the possibility of human cloning in recent years. Despite many objections, this is not only a possibility but also a reality. Human cloning is a scientific revolution. However, it also introduces the potential for physical and psychosocial harm to human beings. From this point of view, it raises profound ethical, social and health related concerns. Human cloning would have an impact on the practice of nursing because it could result in the creation of new physiological and psychosocial conditions that would require nursing care. The nursing profession must therefore evaluate the ethics of human cloning, in particular the potential role of nurses. This article reviews the ethical considerations of reproductive human cloning, discusses the main reasons for concern, and reflects a nursing perspective regarding this issue.

  15. Human somatic cell nuclear transfer and cloning.

    Science.gov (United States)

    2012-10-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer (cloning)," last published in Fertil Steril 2000;74:873-6. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. The pros and cons of human therapeutic cloning in the public debate.

    Science.gov (United States)

    Nippert, Irmgard

    2002-09-11

    Few issues linked to genetic research have raised as much controversial debate as the use of somatic cell nuclear transfer technology to create embryos specifically for stem cell research. Whereas European countries unanimously agree that reproductive cloning should be prohibited there is no agreement to be found on whether or not research into therapeutic cloning should be permitted. Since the UK took the lead and voted in favour of regulations allowing therapeutic cloning the public debate has intensified on the Continent. This debate reflects the wide spectrum of diverse religious and secular moralities that are prevalent in modern multicultural European democratic societies. Arguments range from putting forward strictly utilitarian views that weight the moral issues involved against the potential benefits that embryonic stem cell research may harbour to considering the embryo as a human being, endowed with human dignity and human rights from the moment of its creation, concluding that its use for research is unethical and should be strictly prohibited. Given the current state of dissension among the various European states, it is difficult to predict whether 'non-harmonisation' will prevail or whether in the long run 'harmonisation' of legislation that will allow stem cell research will evolve in the EU.

  17. [The discrete horror of cloning].

    Science.gov (United States)

    Guibourg, Ricardo A

    2009-01-01

    The author raises the topic of cloning after the decision of the Argentine government, which concerned for the "dignity of the human person", passed a decree of need and urgency, No. 200/97 (Annex), prohibiting cloning experiments with human beings. Therefore, considering that the topic is so terribly urgent and necessary, the author feels it is timely to consider it.

  18. Human cloning: category, dignity, and the role of bioethics.

    Science.gov (United States)

    Shuster, Evelyne

    2003-10-01

    Human cloning has been simultaneously a running joke for massive worldwide publicity of fringe groups like the Raelians, and the core issue of an international movement at the United Nations in support of a treaty to ban the use of cloning techniques to produce a child (so called reproductive cloning). Yet, even though debates on human cloning have greatly increased since the birth of Dolly, the clone sheep, in 1997, we continue to wonder whether cloning is after all any different from other methods of medically assisted reproduction, and what exactly makes cloning an 'affront to the dignity of humans.' Categories we adopt matter mightily as they inform but can also misinform and lead to mistaken and unproductive decisions. And thus bioethicists have a responsibility to ensure that the proper categories are used in the cloning debates and denounce those who try to win the ethical debate through well-crafted labels rather than well-reasoned argumentations. But it is as important for bioethicists to take a position on broad issues such as human cloning and species altering interventions. One 'natural question' would be, for example, should there be an international treaty to ban human reproductive cloning?

  19. Keeping up with the cloneses--issues in human cloning.

    Science.gov (United States)

    Rollin, B E

    1999-01-01

    The advent of cloning animals has created a maelstrom of social concern about the "ethical issues" associated with the possibility of cloning humans. When the "ethical concerns" are clearly examined, however, many of them turn out to be less matters of rational ethics than knee-jerk emotion, religious bias, or fear of that which is not understood. Three categories of real and spurious ethical concerns are presented and discussed: 1) that cloning is intrinsically wrong, 2) that cloning must lead to bad consequences, and 3) that cloning harms the organism generated. The need for a rational ethical framework for discussing biotechnological advances is presented and defended.

  20. Telomeres and the ethics of human cloning.

    Science.gov (United States)

    Allhoff, Fritz

    2004-01-01

    In search of a potential problem with cloning, I investigate the phenomenon of telomere shortening which is caused by cell replication; clones created from somatic cells will have shortened telomeres and therefore reach a state of senescence more rapidly. While genetic intervention might fix this problem at some point in the future, I ask whether, absent technological advances, this biological phenomenon undermines the moral permissibility of cloning.

  1. Human cloning: Eastern Mediterranean Region perspective.

    Science.gov (United States)

    Abdur Rab, M; Khayat, M H

    2006-01-01

    Recent advances in genomics and biotechnology have ushered in a new era in health development. Therapeutic cloning possesses enormous potential for revolutionizing medical and therapeutic techniques. Cloning technology, however, is perceived as having the potential for reproductive cloning, which raises serious ethical and moral concerns. It is important that the Islamic countries come to a consensus on this vital issue. Developing science and technology for better health is a religious and moral obligation. There is an urgent need for Muslim scholars to discuss the issue of stem cell research and cloning rationally; such dialogue will not only consider the scientific merits but also the moral, ethical and legal implications.

  2. Procreative liberty, enhancement and commodification in the human cloning debate.

    Science.gov (United States)

    Shapshay, Sandra

    2012-12-01

    The aim of this paper is to scrutinize a contemporary standoff in the American debate over the moral permissibility of human reproductive cloning in its prospective use as a eugenic enhancement technology. I shall argue that there is some significant and under-appreciated common ground between the defenders and opponents of human cloning. Champions of the moral and legal permissibility of cloning support the technology based on the right to procreative liberty provided it were to become as safe as in vitro fertilization and that it be used only by adults who seek to rear their clone children. However, even champions of procreative liberty oppose the commodification of cloned embryos, and, by extension, the resulting commodification of the cloned children who would be produced via such embryos. I suggest that a Kantian moral argument against the use of cloning as an enhancement technology can be shown to be already implicitly accepted to some extent by champions of procreative liberty on the matter of commodification of cloned embryos. It is in this argument against commodification that the most vocal critics of cloning such as Leon Kass and defenders of cloning such as John Robertson can find greater common ground. Thus, I endeavor to advance the debate by revealing a greater degree of moral agreement on some fundamental premises than hitherto recognized.

  3. Towards an understanding of British public attitudes concerning human cloning.

    Science.gov (United States)

    Shepherd, Richard; Barnett, Julie; Cooper, Helen; Coyle, Adrian; Moran-Ellis, Jo; Senior, Victoria; Walton, Chris

    2007-07-01

    The ability of scientists to apply cloning technology to humans has provoked public discussion and media coverage. The present paper reports on a series of studies examining public attitudes to human cloning in the UK, bringing together a range of quantitative and qualitative methods to address this question. These included a nationally representative survey, an experimental vignette study, focus groups and analyses of media coverage. Overall the research presents a complex picture of attitude to and constructions of human cloning. In all of the analyses, therapeutic cloning was viewed more favourably than reproductive cloning. However, while participants in the focus groups were generally negative about both forms of cloning, and this was also reflected in the media analyses, quantitative results showed more positive responses. In the quantitative research, therapeutic cloning was generally accepted when the benefits of such procedures were clear, and although reproductive cloning was less accepted there was still substantial support. Participants in the focus groups only differentiated between therapeutic and reproductive cloning after the issue of therapeutic cloning was explicitly raised; initially they saw cloning as being reproductive cloning and saw no real benefits. Attitudes were shown to be associated with underlying values associated with scientific progress rather than with age, gender or education, and although there were a few differences in the quantitative data based on religious affiliation, these tended to be small effects. Likewise in the focus groups there was little direct appeal to religion, but the main themes were 'interfering with nature' and the 'status of the embryo', with the latter being used more effectively to try to close down further discussion. In general there was a close correspondence between the media analysis and focus group responses, possibly demonstrating the importance of media as a resource, or that the media reflect

  4. Human reproductive cloning: an analysis of the Andrews Report.

    Science.gov (United States)

    Little, Kim

    2002-01-01

    There is nothing like an overwhelming consensus of opinion to encourage a less than rigourous approach to analyzing complex ethical issues. Unfortunately, this is nowhere more apparent than in the discussion of human reproductive cloning contained in the federal House of Representatives Standing Committee on Legal and Constitutional Affairs' report on human cloning, released last August. The report may well fulfil the first half of its project, namely the empirical task of adequately summarizing and categorizing the various submissions made to the Committee. However, it is clearly inadequate as a discussion of the ethical and legal permissibility of human reproductive cloning.

  5. A review of Greek law on human cloning.

    Science.gov (United States)

    Mavroforou, Anna; Giannoukas, Athanasios; Michalodimitrakis, Emmanuel

    2003-01-01

    The creation of Dolly, a cloned lamb from adult cells was a major scientific breakthrough, which opened new avenues for many research fields such as reproductive medicine, transplantation and biotechnology. However this achievement brought to public attention the theoretical possibility of human reproductive cloning. Inevitably heated debate occurred on several ethical and legal consequences of the prospect of human cloning. At the present time there is no legal framework in any country to respond to this challenge in a pragmatic way in order to protect human rights and at the same time to allow science to work for the best interests of mankind. Greece is a European Union country with its own traditions, history, culture and beliefs but without political and legislative experience in the handling of medical and biotechnological matters. This paper aims to discuss the legal issues likely to be raised by the prospect of human reproductive cloning in relation to the current state of the Greek legal system.

  6. Evidence for a human-specific Escherichia coli clone.

    Science.gov (United States)

    Clermont, Olivier; Lescat, Mathilde; O'Brien, Claire L; Gordon, David M; Tenaillon, Olivier; Denamur, Erick

    2008-04-01

    Escherichia coli is a widespread commensal of the vertebrate intestinal tract. Until recently, no strong association between a particular clone and a given host species has been found. However, members of the B2 subgroup VIII clone with an O81 serotype appear to be human host specific. To determine the degree of host specificity exhibited by this clone, a PCR-based assay was used to screen 723 faecal and clinical isolates from humans, and 904 faecal isolates from animals. This clone was not detected among the animal isolates, but was discovered in people living in Africa, Europe and South America. The clone is rarely isolated from people suffering from intestinal or extraintestinal disease and is avirulent in a mouse model of extraintestinal infection. Fine-scale epidemiological analysis suggests that this clone is competitively dominant relative to other members of the B2 phylogenetic group and that it has increased in frequency over the past 20 years. This clone appears to be a good candidate for use as a probiotic, and may be suitable as an indicator of human faecal contamination in microbial source tracking studies.

  7. Analysis of an epigenetic argument against human reproductive cloning.

    Science.gov (United States)

    Nordgren, Anders

    2006-08-01

    Human reproductive cloning is a much disputed ethical issue. This technology is often condemned as being contrary to human dignity. However, there are also risk arguments. An ethical argument that is often put forward by scientists but seldom developed in more detail focuses on health risks in animal cloning. There is a high risk that animal clones exhibit abnormalities and these are increasingly believed to be due to errors in epigenetic reprogramming. The argument is that human reproductive cloning should not be carried out because human clones are also likely to exhibit abnormalities due to inappropriate epigenetic reprogramming. Different versions of this epigenetic argument are analysed, a categorical version and a non-categorical. The non-categorical version is suggested to be more well-considered. With regard to policy making on human reproductive cloning, the categorical version can be used to prescribe a permanent ban, while the non-categorical version can be used to prescribe a temporary ban. The implications of the precautionary principle--as interpreted in the European Union--are investigated. The conclusion is that it seems possible to support a temporary ban by reference to this principle.

  8. [Cloning and law in Hungary].

    Science.gov (United States)

    Julesz, Máté

    2015-03-01

    Reproductive human cloning is prohibited in Hungary, as in many other countries. Therapeutic human cloning is not prohibited, just like in many other countries. Stem cell therapy is also allowed. Article III, paragraph (3) of the Hungarian basic law (constitution) strictly forbids total human cloning. Article 1 of the Additional Protocol to the Oviedo Convention, on the Prohibition of Cloning Human Beings (1998) stipulates that any intervention seeking to create a human being genetically identical to another human being, whether living or dead, is prohibited. In Hungary, according to Article 174 of the Criminal Code, total human cloning constitutes a crime. Article 180, paragraph (3) of the Hungarian Act on Health declares that embryos shall not be brought about for research purposes; research shall be conducted only on embryos brought about for reproductive purposes when this is authorized by the persons entitled to decide upon its disposal, or when the embryo is damaged. Article 180, paragraph (5) of the Hungarian Act on Health stipulates that multiple individuals who genetically conform to one another shall not be brought about. According to Article 181, paragraph (1) of the Hungarian Act on Health, an embryo used for research shall be kept alive for not longer than 14 days, not counting the time it was frozen for storage and the time period of research.

  9. Cloning and characterization of the neural isoforms of human dystonin

    Energy Technology Data Exchange (ETDEWEB)

    Brown, A.; Dalpe, G.; Mathieu, M.; Kothary, R. [Univ. of Montreal, Quebec (Canada)

    1995-10-10

    Dystonia musculorum (dt) is a hereditary neurodegenerative disease in mice that leads to a sensory ataxia. We have identified and cloned a gene encoded at the dt locus. The product of the dt gene, dystonin, is a neural isoform of a hemidesmosomal protein bullous pemphigoid antigen 1 (bpag1). To investigate the potential role of dystonin in human neuropathies, we have cloned the neural-specific 5{prime} exons of the human DT gene that together with the previously cloned BPAG1 sequences comprise human dystonin. The mouse and human dystonin genes demonstrate the same spectrum of alternatively spliced products, and the amino acid sequences of the neural-specific exons in the mouse and human genes are over 96% identical. 17 refs., 2 figs.

  10. Should we clone human beings? Cloning as a source of tissue for transplantation.

    Science.gov (United States)

    Savulescu, J

    1999-01-01

    The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus. PMID:10226910

  11. Should we clone human beings? Cloning as a source of tissue for transplantation.

    Science.gov (United States)

    Savulescu, J

    1999-04-01

    The most publicly justifiable application of human cloning, if there is one at all, is to provide self-compatible cells or tissues for medical use, especially transplantation. Some have argued that this raises no new ethical issues above those raised by any form of embryo experimentation. I argue that this research is less morally problematic than other embryo research. Indeed, it is not merely morally permissible but morally required that we employ cloning to produce embryos or fetuses for the sake of providing cells, tissues or even organs for therapy, followed by abortion of the embryo or fetus.

  12. [The status of human cloning in the international setting].

    Science.gov (United States)

    Rey del Castillo, Javier

    2006-01-01

    The General Assembly of the United Nations submitted a Declaration on Human Cloning in March 2005. The text of such Declaration was the result of a difficult and long process, taking more than three years. Being a Declaration instead of a Resolution, it has not legal capability in inforcing United Nations members to act according to its recommendations. This article begins with an explanation of several terms referred to cloning. Different countries' legislation on cloning is analyzed. Positions of the same countries at the Convention of the United Nations are as well analyzed. Comparing both countries' views shows that national legislation on cloning is independent and orientated by some countries' particular interests and biological and ethical views on these issues. Future developments on human cloning and its applications will be shared among all countries, both the ones currently allowing and supporting "therapeutic" cloning and the ones now banning it. In such case, it would be important to reach agreements on these issues at an international level. The article discusses possible legislative developments and offers some proposals to reach such agreements.

  13. The ethics of cloning and human embryo research.

    Science.gov (United States)

    Saran, Madeleine

    2002-01-01

    The successful cloning experiments that led to Dolly in 1997 have raised many ethical and policy questions. This paper will focus on cloning research in human embryonic cells. The possible gains of the research will be judged against the moral issues of doing research on a person. This paper concludes that while the embryo has some moral status, its moral status is outweighed by the multitude of benefits that embryonic stem cell research will bring to humanity. Policy suggestions are given for dealing with this new and developing field of stem cell research.

  14. 40 CFR 26.1704 - Prohibition of reliance on unethical human research with non-pregnant, non-nursing adults...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Prohibition of reliance on unethical human research with non-pregnant, non-nursing adults conducted before April 7, 2006. 26.1704 Section 26... Prohibition of reliance on unethical human research with non-pregnant, non-nursing adults conducted...

  15. 40 CFR 26.1705 - Prohibition of reliance on unethical human research with non-pregnant, non-nursing adults...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Prohibition of reliance on unethical human research with non-pregnant, non-nursing adults conducted after April 7, 2006. 26.1705 Section 26... Prohibition of reliance on unethical human research with non-pregnant, non-nursing adults conducted...

  16. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    Science.gov (United States)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  17. Psychological aspects of human cloning and genetic manipulation: the identity and uniqueness of human beings.

    Science.gov (United States)

    Morales, N M

    2009-01-01

    Human cloning has become one of the most controversial debates about reproduction in Western civilization. Human cloning represents asexual reproduction, but the critics of human cloning argue that the result of cloning is not a new individual who is genetically unique. There is also awareness in the scientific community, including the medical community, that human cloning and the creation of clones are inevitable. Psychology and other social sciences, together with the natural sciences, will need to find ways to help the healthcare system, to be prepared to face the new challenges introduced by the techniques of human cloning. One of those challenges is to help the healthcare system to find specific standards of behaviour that could be used to help potential parents to interact properly with cloned babies or children created through genetic manipulation. In this paper, the concepts of personality, identity and uniqueness are discussed in relationship to the contribution of twin studies in these areas. The author argues that an individual created by human cloning techniques or any other type of genetic manipulation will not show the donor's characteristics to the extent of compromising uniqueness. Therefore, claims to such an effect are needlessly alarmist.

  18. Scientific and medical aspects of human reproductive cloning

    National Research Council Canada - National Science Library

    Committee on Science

    2002-01-01

    ...), and the Institute of Medicine (IOM). It is a result of work done by the Panel on Scientific and Medical Aspects of Human Cloning, a joint panel of the Committee on Science, Engineering, and Public Policy (COSEPUP) and the Board on Life Sciences (BLS). The members of the panel responsible for the report were chosen for their special competences and with regard ...

  19. Molecular cloning of the human excision repair gene ERCC-6.

    NARCIS (Netherlands)

    C. Troelstra (Christine); H. Odijk (Hanny); J. de Wit (Jan); A. Westerveld (Andries); L.H. Thompson; D. Bootsma (Dirk); J.H.J. Hoeijmakers (Jan)

    1990-01-01

    textabstractThe UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5

  20. Reproductive cloning in humans and therapeutic cloning in primates: is the ethical debate catching up with the recent scientific advances?

    Science.gov (United States)

    Camporesi, S; Bortolotti, L

    2008-09-01

    After years of failure, in November 2007 primate embryonic stem cells were derived by somatic cellular nuclear transfer, also known as therapeutic cloning. The first embryo transfer for human reproductive cloning purposes was also attempted in 2006, albeit with negative results. These two events force us to think carefully about the possibility of human cloning which is now much closer to becoming a reality. In this paper we tackle this issue from two sides, first summarising what scientists have achieved so far, then discussing some of the ethical arguments in favour and against human cloning which are debated in the context of policy making and public consultation. Therapeutic cloning as a means to improve and save lives has uncontroversial moral value. As to human reproductive cloning, we consider and assess some common objections and failing to see them as conclusive. We do recognise, though, that there will be problems at the level of policy and regulation that might either impair the implementation of human reproductive cloning or make its accessibility restricted in a way that could become difficult to justify on moral grounds. We suggest using the time still available before human reproductive cloning is attempted successfully to create policies and institutions that can offer clear directives on its legitimate applications on the basis of solid arguments, coherent moral principles, and extensive public consultation.

  1. Patenting humans: clones, chimeras, and biological artifacts.

    Science.gov (United States)

    Hurlbut, William B

    2005-01-01

    The momentum of advances in biology is evident in the history of patents on life forms. As we proceed forward with greater understanding and technological control of developmental biology there will be many new and challenging dilemmas related to patenting of human parts and partial trajectories of human development. These dilemmas are already evident in the current conflict over the moral status of the early human embryo. In this essay, recent evidence from embryological studies is considered and the unbroken continuity of organismal development initiated at fertilization is asserted as clear and reasonable grounds for moral standing. Within this frame of analysis, it is proposed that through a technique of Altered Nuclear Transfer, non-organismal entities might be created from which embryonic stem cells could be morally procured. Criteria for patenting of such non-organismal entities are considered.

  2. Cloning and expression of special F protein from human liver

    Institute of Scientific and Technical Information of China (English)

    Shu-Ye Liu; Xin-Da Yu; Chun-Juan Song; Wei Lu; Jian-Dong Zhang; Xin-Rong Shi; Ying Duan; Ju Zhang

    2007-01-01

    AIM:To clone human liver special F protein and to express it in a prokaryotic system.METHODS:Total RNA was isolated from human liver tissue and first-strand cDNA was reverse transcribed using the PCR reverse primer. Following this,cDNA of the F protein was ligated into the clone vector pUCm-T. The segment of F protein's cDNA was subcloned into the expression vector pET-15b and transformed into E coli BL21 (DEB) pLyss. Isopropy-β-D-thiogalactoside (IPTG) was then used to induce expression of the target protein.RESULTS:The cDNA clone of human liver special F protein (1134bp) was successfully produced,with the cDNA sequence being published in Gene-bank:DQ188836. We confirmed the expression of F protein by Western blot with a molecular weight of 43 kDa. The expressed protein accounted for 40% of the total protein extracted.CONCLUSION:F protein expresses cDNA clone in a proKaryotic system,which offers a relatively simple way of producing sufficient quantities of F protein and contributes to understanding the principal biological functions of this protein.

  3. 40 CFR 26.1703 - Prohibition of reliance on research involving intentional exposure of human subjects who are...

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 1 2010-07-01 2010-07-01 false Prohibition of reliance on research...), nursing women, or children. 26.1703 Section 26.1703 Protection of Environment ENVIRONMENTAL PROTECTION... Results of Human Research in EPA Actions § 26.1703 Prohibition of reliance on research...

  4. Human Cloning as the Other in Ishiguro's Never Let Me Go

    National Research Council Canada - National Science Library

    Guo, Wen

    2015-01-01

    In her article "Human Cloning as the Other in Ishiguro's Never Let Me Go" Wen Guo analyzes Kazuo Ishiguro's novel with focus on Ishiguro's analogy between human cloning and people of marginality in contemporary society...

  5. Cloning of Integral Mature Peptide Gene of Human GDF-5

    Institute of Scientific and Technical Information of China (English)

    王万山; 顾为望; 王启伟; 朴仲贤; 朴英杰

    2004-01-01

    Summary: The integral mature peptide gene of human growth differentiation factor-5 (GDF-5) was cloned to provide the essential foundation for study on the biological characteristics of GDF-5 at gene and protein levels. Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR methods from the total RNA extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T-hGDF-5 was analyzed by sequence analysis. DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Our results showed that the integral mature peptide gene of human GDF-5 was cloned successfully from human fetal cartilage tissue, and totally identified with the sequence of human GDF-5 in Genbank.

  6. Human cloning laws, human dignity and the poverty of the policy making dialogue.

    Science.gov (United States)

    Caulfield, Timothy

    2003-07-29

    The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. The author critiques one of the most commonly used ethical justifications for cloning laws - the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning. It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

  7. Human cloning laws, human dignity and the poverty of the policy making dialogue

    Directory of Open Access Journals (Sweden)

    Caulfield Timothy

    2003-07-01

    Full Text Available Abstract Background The regulation of human cloning continues to be a significant national and international policy issue. Despite years of intense academic and public debate, there is little clarity as to the philosophical foundations for many of the emerging policy choices. The notion of "human dignity" is commonly used to justify cloning laws. The basis for this justification is that reproductive human cloning necessarily infringes notions of human dignity. Discussion The author critiques one of the most commonly used ethical justifications for cloning laws – the idea that reproductive cloning necessarily infringes notions of human dignity. He points out that there is, in fact, little consensus on point and that the counter arguments are rarely reflected in formal policy. Rarely do domestic or international instruments provide an operational definition of human dignity and there is rarely an explanation of how, exactly, dignity is infringed in the context reproductive cloning. Summary It is the author's position that the lack of thoughtful analysis of the role of human dignity hurts the broader public debate about reproductive cloning, trivializes the value of human dignity as a normative principle and makes it nearly impossible to critique the actual justifications behind many of the proposed policies.

  8. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  9. The ethics of human reproductive cloning: when world views collide.

    Science.gov (United States)

    Cohen, Cynthia B

    2004-01-01

    Two camps in bioethics with seemingly opposing world views have staked out conflicting positions regarding the ethics of human reproductive cloning. These camps do not appear to share common concepts or ways of reasoning through which to exchange views and come to a meeting of minds about uses of this technology. Yet analysis of their respective approaches to several issues surrounding reproductive cloning, such as where the ethical limits of individual reproductive choice lie, whether the use of this technology would violate human dignity, whether it would create risks to the resulting fetuses and children that would make its use intolerable, and whether it would challenge certain core social values, reveals that they are not wholly opposed to one another. Indeed, it displays that they hold certain beliefs, values, and concerns in common. Moreover, it indicates that the different world views that they each presuppose, while flawed in certain respects, do not collide in every respect, but can be reconciled in significant ways that provide fertile ground for agreement about several issues related to human reproductive cloning.

  10. The regulation of science and the Charter of Rights: would a ban on non-reproductive human cloning unjustifiably violate freedom of expression?

    Science.gov (United States)

    Billingsley, Barbara; Caulfield, Timothy

    2004-01-01

    Non-Reproductive Human Cloning (NRHC) allows researchers to develop and clone cells, including non-reproductive cells, and to research the etiology and transmission of disease. The ability to clone specific stem cells may also allow researchers to clone cells with genetic defects and analyze those cells with more precisions. Despite those potential benefits, Parliament has banned such cloning due to a myriad of social and ethical concerns. In May 2002, the Canadian Government introduced Bill C-13 on assisted human reproductive technologies. Bill C-13 deals with both the scientific and the clinical use of human reproductive materials, and it prohibits a number of other activities, including NRHC. Although the Supreme Court of Canada has never ruled on whether scientific experiments area form of expression, academic support exists for this notion. The authors go through the legal analysis that would be required to find that scientific experiments are expression, focusing in part on whether NRHC could be considered violent and thus fall outside the protection of section 2(b). The latter question is complicated by the ongoing policy debate over whether an "embryonic cell" is property of human life. The authors then consider whether a ban on NRHC could be justified under section 1 of the Charter. They conclude that both the breadth of the legislative purpose and the proportionality of the measure are problematic. Proportionality is a specific concern because the ban could be viewed as an outright denial of scientific freedom of expression. Although consistent with current jurisprudence on freedom of expression, this paper runs against the flow of government policy in the areas of regulation and prohibition of non-reproductive human cloning. As there has been no Charter litigation to date on whether scientific research is a form of expression, the authors introduce a new way of looking at the legality of the regulation of new reproductive technologies.

  11. Human estrogen sulfotransferase gene (STE): Cloning, structure, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Aksoy, I.A.; Weinshilboum, M. [Mayo Foundation, Rochester, MI (United States)] [and others

    1995-09-01

    Sulfation is an important pathway in the metabolism of estrogens. We recently cloned a human liver estrogen sulfotransferase (EST) cDNA. We have now determined the structure and chromosomal localization of the EST gene, STE, as a step toward molecular genetic studies of the regulation of EST in humans. STE spans approximately 20 kb and consists of 8 exons, ranging in length from 95 to 181 bp. The locations of most exon-intron splice junctions within STE are identical to those found in a human phenol ST (PST) gene, STM, and in a rat PST gene. In addition, the locations of five STE introns are also conserved in the human dehydroepiandrosterone (DBEA) ST gene, STD. The 5{prime} flanking region of STE contains one CCAAT and two TATA sequences. The location of one of the TATA box elements is in excellent agreement with the site of transcription initiation as determined by 5{prime}-rapid amplification of cDNA ends. STE was mapped to human chromosome 4q13.1 by fluorescence in situ hybridization. Cloning and structural characterization of STE will now make it possible to study potential molecular genetic mechanisms involved in the regulation of EST in human tissues. 50 refs., 6 figs., 1 tab.

  12. Annual banned-substance review: the Prohibited List 2008-analytical approaches in human sports drug testing.

    Science.gov (United States)

    Thevis, Mario; Kuuranne, Tiia; Geyer, Hans; Schänzer, Wilhelm

    2009-01-01

    The list of prohibited substances and methods of doping issued by the World Anti-Doping Agency is updated and modified annually based on most recent developments and scientific data. Compounds and methods are maintained, added, or removed from the list, or they are placed in so-called monitoring programmes that have been established to obtain reliable data on the prevalence of particular substances and methods in- and/or out-of-competition. Consequently, doping control laboratories continuously update, modify and optimize existing screening and confirmation assays to ensure utmost comprehensiveness in detecting the prohibited and monitored substances as well as chemically and pharmacologically related analogs. The annual banned-substance review for human sports drug testing critically summarizes recent innovations in analytical approaches supporting the detection of established and newly outlawed substances and methods of doping. Literature from January 2007 through September 2008 as indexed in Medline and Web of Science was screened and articles on detection methods for substances and methods of doping in humans were compiled according to the 2008 Prohibited List of the World Anti-Doping Agency. Few new approaches were presented for individual doping agents and the majority of reports demonstrated new options for increasing the comprehensiveness of existing doping control assays. In addition, new techniques in separation and/or ionization of analytes complementary to commonly used procedures were described, which, so far, did not meet all requirements of sports drug testing. (c) 2009 John Wiley & Sons, Ltd.

  13. The Dao of human cloning: utopian/dystopian hype in the British press and popular films.

    Science.gov (United States)

    Jensen, Eric

    2008-04-01

    The issue of human cloning has featured in the national science policy agendas in both the United States and the United Kingdom since the announcement in 1997 of Dolly the cloned sheep's birth in Scotland. Such news stories suggesting the imminent cloning of humans have inspired fictional entertainment media over the years, including numerous popular films. Study 1 examines elite British press coverage of human cloning from 1997 to 2004 (n = 857). Study 2 focuses on five human cloning films released between 1978 and 2003. Two sharply divergent discourses emerged from these data. Unqualified hope and habitually hyped claims of future cures permeated the press discourse. In contrast, the films constructed human cloning as an inherently dangerous technology often wielded by hubristic scientists in the tradition of Frankenstein. Both the predominately positive hype in the broadsheet press and the largely negative hype in the films indicate an impoverished and "thin" public debate on the issue of human cloning.

  14. Cloning and expression of human colon mast cell carboxypeptidase

    Institute of Scientific and Technical Information of China (English)

    Zhang-Quan Chen; Shao-Heng He

    2004-01-01

    AIM: To clone and express the human colon mast cell METHODS: Total RNA was extracted from colon tissue, and the cDNA encoding human colon mast cell carboxypeptidase was amplified by reverse-transcription PCR (RT-PCR). The product cDNA was subcloned into the prokaryotic expression vector pMAL-c2x and eukaryotic expression vector pPIC9K to conrtruct prokaryotic expression vector pMAL/human MC-CP (hMC-CP) and eukaryotic pPIC9K/hMC-CP. The recombinant fusion protein expressed in E.coli was induced with IPTG and purified by amylose affinity chromatography. After digestion with factor Xa, recombinant hMC-CP was purified by heparin agarose chromatography. The recombinant hMC-CP expressed in Pichia pastoris (P.pastoris) was induced with methanol and analyzed by SDS-PAGE, Western blot, N-terminal amino acid RESULTS: The cDNA encoding the human colon mast cell carboxypeptidase was cloned, which had five nucleotide variations compared with skin MC-CP cDNA. The recombinant hMC-CP protein expressed in E.coli was purified with amylose affinity chromatography and heparin agarose chromatogphy.SDS-PAGE and Western blot analysis showed that the recombinant protein expressed by E. coli had a molecular weight of 36 kDa and reacted to the anti-native hMC-CP monoclonal antibody (CA5). The N-terminal amino acid sequence confirmed further the product was hMC-CP. E. coli generated hMC-CP showed a very low level of enzymatic activity, but P. pastoris produced hMC-CP had a relatively high enzymatic activity towards a synthetic substrate hippuryl-L-phenylalanine.carboxypeptidase can be successfully cloned and expressed in E.coli and P. pastoris, which will contribute greatly to the fonctional study on hMC-CP.

  15. ES cells derived from cloned embryos in monkey - a jump toward human therapeutic cloning

    Institute of Scientific and Technical Information of China (English)

    Xiangzhong Yang; Sadie L Smith

    2007-01-01

    @@ Therapeutic cloning refers to the derivation of embryonic stem cells (ntESC) from embryos derived from somatic cell nuclear transfer (SCNT) also known as cloning. Cloning involves transplanting a differentiated cell into an oocyte that has had its nucleus (DNA) removed.

  16. Cloning

    Science.gov (United States)

    ... sheep that have been genetically modified to produce milk that contains a human protein essential for blood clotting. The hope is that someday this protein can be purified from the milk and given to humans whose blood does not ...

  17. Demographic profile of states with human cloning laws: morality policy meets political economy.

    Science.gov (United States)

    Stabile, Bonnie

    2007-03-01

    This analysis seeks to identify factors that may shape the policy stance - whether restrictive or permissive - that each state in the United States with a human cloning law in place takes toward human therapeutic cloning. The investigation also considers if cloning policy is more the product of morality politics or political economy. Results show that among states with human cloning policies in place, those with a greater biotechnological capacity, more permissive abortion laws, fewer Evangelical Protestants, and higher political liberalism rankings are more likely to have permissive cloning laws. A higher Roman Catholic population is strongly associated with permissive cloning laws, rather than restrictive cloning laws as originally supposed. Factors with morality policy and economic bases were both found to be associated with cloning policy outcomes. Results suggest that morality policies, though distinct in some ways, do share determinants with public policies based on political economy.

  18. 40 CFR 26.1203 - Prohibition of research involving intentional exposure of any human subject who is a pregnant...

    Science.gov (United States)

    2010-07-01

    ... intentional exposure of any human subject who is a pregnant woman (and therefore her fetus), a nursing woman... Exposure of Human Subjects who are Children or Pregnant or Nursing Women § 26.1203 Prohibition of research involving intentional exposure of any human subject who is a pregnant woman (and therefore her fetus),...

  19. CLONING AND SEQUENCING OF MATURED FRAGMENT OF HUMAN NEVER GROWTH FACTOR GENE

    Institute of Scientific and Technical Information of China (English)

    马巍; 吴玲; 王德利; 刘淼; 任惠民; 杨广笑; 王全颖

    2003-01-01

    Objective Molecular cloning and sequencing of the human matured fragment of human nerve growth factor(NGF) gene. Methods Extracting the human genomic DNA from the white blood cells as templates, the gene of NGF was cloned by using PCR and T-vector cloning method. Screening the positive clones and identified by the restriction enzymes, and then the cloned amplified fragment was sequenced and analyzed. Results DNA sequence comparison the cloned gene of NGF with the GenBank (V01511) sequence demonstrated that both of sequences were identical, 354bp length. Conclusion Cloning the NGF gene from the human genomic DNA has paved the way for further study on gene therapy of nerve system injury.

  20. Reproductive cloning and human health: an ethical, international, and nursing perspective.

    Science.gov (United States)

    Sanchez-Sweatman, L R

    2000-03-01

    Human reproductive cloning came to the public's attention when Dolly, a sheep, was cloned in Scotland in 1997. This news quickly spread around the world causing both excitement at the possibilities that cloning techniques could offer, as well as apprehension about the ethical, social and legal implications should human reproductive cloning become possible. Many international organizations, such as the World Health Organization, the International Council of Nurses, and governments were concerned about the impact of human reproductive cloning on human health, dignity and human rights. To this end, many institutions have drafted resolutions, protocols and position statements outlining their concerns. This paper will outline some of the major ethical issues surrounding human reproductive cloning, the position of various international organizations and governments, and specifically the position of the International Council of Nurses.

  1. Cloning and characterization of a functional human ¿-aminobutyric acid (GABA) transporter, human GAT-2

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Meinild, Anne-Kristine; Jensen, Anders A.

    2007-01-01

    Plasma membrane gamma-aminobutyric acid (GABA) transporters act to terminate GABA neurotransmission in the mammalian brain. Intriguingly four distinct GABA transporters have been cloned from rat and mouse, whereas only three functional homologs of these transporters have been cloned from human....... The aim of this study therefore was to search for this fourth missing human transporter. Using a bioinformatics approach, we successfully identified and cloned the full-length cDNA of a so far uncharacterized human GABA transporter (GAT). The predicted protein displays high sequence similarity to rat GAT......-2 and mouse GAT3, and in accordance with the nomenclature for rat GABA transporters, we therefore refer to the transporter as human GAT-2. We used electrophysiological and cell-based methods to demonstrate that this protein is a functional transporter of GABA. The transport was saturable...

  2. Ethical attitudes on human cloning among professionals in Taiwan and the policy implications for regulation.

    Science.gov (United States)

    Yang, Che-Ming; Chung, Chun-Chih; Lu, Meei-Shiow; Lin, Chiou-Fen; Chen, Jiun-Shyan

    2005-01-01

    This research focused on understanding the attitudes toward human cloning in Taiwan among professionals in healthcare, law, and religion. The study was conducted utilizing a structured questionnaire. 220 healthcare professionals from two regional hospitals located in Taipei, 351 religious professionals in the northern Taiwan and 711 legal professionals were selected by to receive questionnaires. The valid response rate is 42.1% The questions were generated by an expert panel and represented major arguments in the human cloning debate. There were a total of six Likert scaled questions in the questionnaire. The responses were coded from 1 to 5 with 1 representing strong opposition to human cloning, 3 representing a neutral attitude; and 5 representing a strong favorable attitude toward human cloning. Healthcare professionals had the highest overall average score of 2.14 and the religious professionals had the lowest average at 1.58. All three categories of respondents' attitude toward cloning ranged from mild opposition to strong opposition to human cloning. The religious professionals were more strongly opposed to cloning. Age, education, and religion significantly influenced attitudes toward cloning. Professionals between fifty-one and sixty years old, those with less education, and Roman Catholic professionals were more strongly opposed to cloning. Religious professionals were more strongly opposed to human cloning than professionals in healthcare or law. Younger professionals as an age group demonstrated less opposition to human cloning. Regulation of human cloning will be influenced by professionals in healthcare, law, and religion, and the regulatory environment chosen now will play a pivotal role in influencing the acceptance of human cloning in the future.

  3. Cloning the human gene for macrophage migration inhibitory factor (MIF)

    Energy Technology Data Exchange (ETDEWEB)

    Paralkar, V.; Wistow, G. (National Institutes of Health, Bethesda, MD (United States))

    1994-01-01

    Macrophage migration inhibitory factor (MIF) was originally identified as a lymphokine. However, recent work strongly suggests a wider role for MIF beyond the immune system. It is expressed specifically in the differentiating cells of the immunologically privileged eye lens and brain, is a delayed early response gene in fibroblasts, and is expressed in many tissues. Here, the authors report the structure of the remarkably small gene for human MIF that has three exons separated by introns of only 189 and 95 bp and covers less than 1 kb. The cloned sequence also includes 1 kb of 5[prime] flanking region. Primer extension and 5[prime] rapid amplification of cDNA ends (RACE) of human brain RNA both indicate the presence of a single transcription start site in a TATA-less promoter. Northern blot analysis shows a single size of MIF mRNA (about 800 nt) in all human tissues examined. In contrast to previous reports, they find no evidence for multiple genes for MIF in the human genome. 20 refs., 3 figs.

  4. Cloning and characterization of the human PAX7 promoter.

    Science.gov (United States)

    Murmann, O V; Niggli, F; Schäfer, B W

    2000-04-01

    PAX7, a member of the PAX transcription factor gene family, is normally expressed at high levels during development in the neural tube and in skeletal muscle precursor cells. Interestingly, PAX7 expression was also identified in tumor cells developing from these cell types. To date not much is known about the molecular mechanisms controlling the regulation of PAX7 expression. Therefore, we have cloned and sequenced part of the proximal 5'-flanking region of the human PAX7 gene. Computer-based sequence analysis identified putative binding sites for basic transcription factors. Analysis of a series of deletion constructs in different cell types suggested that a distal region containing several E-boxes might be involved in muscle-specific expression of PAX7, and that a distinct proximal region can enhance basal PAX7 expression in tumor cells.

  5. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    OpenAIRE

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-01-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its o...

  6. Cloning and characterization of the human USP22 gene promoter.

    Directory of Open Access Journals (Sweden)

    Jianjun Xiong

    Full Text Available Ubiquitin-specific processing enzyme 22 (USP22 plays a direct role in regulating cell cycle, and its overexpression has been reported to be involved in tumor progression. However, little is known about the regulation of USP22 transcription. In this study, we cloned and characterized the human USP22 promoter. Using 5' RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis showed that the sequence between -210 and -7 contains the basal promoter for USP22 in human fibroblast and tumor cells. Surprisingly, mutations in a putative Sp1 binding site immediately upstream of the USP22 transcriptional start site (-13 to -7 resulted in a significant induction of promoter activity. Further study revealed that Sp1 binds to this site in human normal fibroblast cells, and treatment with the Sp1 inhibitor mithramycin A led to a marked increase in USP22 transcript levels. Forced expression of exogenous Sp1 repressed the USP22 promoter activity in HeLa cells. In contrast, knockdown of Sp1 enhanced USP22 promoter activity and mRNA levels. These data suggest that Sp1 is a crucial regulator of USP22 transcription.

  7. Partial Cloning and Nucleotide Sequencing of Glutamate Decarboxylase Gene Isoform 65 from Human Brain

    Directory of Open Access Journals (Sweden)

    Abolghasem Esmaeili

    2015-04-01

    Conclusion: Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

  8. Endomorphins fully activate a cloned human mu opioid receptor.

    Science.gov (United States)

    Gong, J; Strong, J A; Zhang, S; Yue, X; DeHaven, R N; Daubert, J D; Cassel, J A; Yu, G; Mansson, E; Yu, L

    1998-11-13

    Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.

  9. Cloning, expression and location of RNase9 in human epididymis

    Directory of Open Access Journals (Sweden)

    Lin YQ

    2008-11-01

    Full Text Available Abstract Background Mammalian spermatozoa become fully motile and fertile during transit through the luminal fluid of the epididymis. At least 200 proteins are present in the epididymal lumen, but the potential roles of these luminal proteins in male fertility are unknown. Investigation of the function of these proteins will elucidate the mechanism of sperm maturation, and also provide new drug targets for male contraception. We cloned RNase9 from a human epididymis cDNA library for characterization and analysis of its functions. Findings It was predicted that human RNase9 gene was located on chromosome 14q11.2 and encoded a 205 amino acids protein with a signal peptide of 26 amino acids at the N-terminus. The protein had eight conserved cysteine residues characteristic of the RNase A family members and several potential post-translational modification sites. At the transcriptional level, RNase9 was expressed in a wide variety of tissues, and the expression was higher in men than in boys. RNase9 was localized to the post-equatorial region of the sperms' head. Immunofluorescence staining showed that RNase9 protein was present mostly in the epithelium of the epididymal tubule. Recombinant RNase9 had no ribonuclease activity. In addition, RNase9 had no detectable effect on sperm motility and fertilization as demonstrated by blocking spermatozoa with anti-RNase9 polyclonal serum. Conclusion RNase9 is expressed in a wide variety of tissues. It is located on the post-equatorial region of the sperm head and the epithelium of epididymal tubule. Although RNase9 belongs to the RNase A family, it has no ribonuclease activity.

  10. Construction and characterization of human chromosome 2-specific cosmid, fosmid, and PAC clone libraries

    Energy Technology Data Exchange (ETDEWEB)

    Gingrich, J.C.; Boehrer, D.M.; Garnes, J.A. [Lawrence Livermore National Lab., CA (United States)] [and others

    1996-02-15

    This article discusses the construction and characterization of three human chromosome 2-specific clone libraries. A chromosome 2-specific PAC library was also constructed from a hybrid cell line. The chromosome 2 coverage of each of the three libraries was further determined by PCR screening clone pools with 82 chromosome 2-specific STSs. 47 refs., 3 figs., 1 tab.

  11. Just Another Reproductive Technology? The Ethics of Human Reproductive Cloning as an Experimental Medical Procedure

    National Research Council Canada - National Science Library

    D. Elsner

    2006-01-01

    Human reproductive cloning (HRC) has not yet resulted in any live births. There has been widespread condemnation of the practice in both the scientific world and the public sphere, and many countries explicitly outlaw the practice...

  12. A human T cell clone that mediates the monocyte procoagulant response to specific sensitizing antigen.

    Science.gov (United States)

    Schwartz, B S; Reitnauer, P J; Hank, J A; Sondel, P M

    1985-09-01

    A panel of human purified protein derivative of the tubercle bacillus (PPD)-reactive T cell clones was derived by cloning out of soft agar followed by cultivation on inactivated feeder cells in the presence of interleukin-2. 1 of 4 clones tested was able to mediate an increase in monocyte procoagulant activity (PCA) in response to PPD. All four clones had identical surface marker phenotypes (T4+, T8-) and proliferated in response to antigen. The reactive T cell clone possessed no PCA of its own, but upon being presented with PPD was able to instruct monocytes to increase their expression of PCA. Antigen presentation could be performed only by autologous monocytes; allogeneic monocytes from donors unrelated to the donor of the reactive clone could not present antigen to cells of the clone in a way that would initiate the procoagulant response. Cells of the reactive clone did not mediate increased monocyte PCA in response to Candida, even though peripheral blood mononuclear cells from the donor demonstrated increased PCA to both Candida and PPD. Thus, the PCA response to specific antigen can be mediated by a single clone of cells that shows specificity in the recognition of both antigen and antigen presenting cell.

  13. Molecular Cloning of Human Gene(s) Directing the Synthesis of Nervous System Cholinesterases

    Science.gov (United States)

    1987-09-01

    Report No. 4 If MOLECULAR CLONING OF O HUMAN GENE(S) DIRECTING qTHE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASES cc Annual/Final Report 0 N November...62734A I734A875 IAl 451 MOLECULAR CLONING OF HUMAN GEME(S) DIRECTING THE SYNTHESIS OF NERVOUS SYSTEM CHOLINESTERASE 12. PERSONAL AUTHOR(S) Hermona Soreq...important roles in regulating the pace and mode of function of particular types of synapses. For example, molecular cloning of the nicotinic (44-46) and the

  14. Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Sun, W.; Ferrer-Montiel, A.V.; Schinder, A.F.; Montal, M. (Univ. of California, San Diego, La Jolla (United States)); McPherson, J.P. (Univ. of California, Irvine (United States)); Evans, G.A. (Salk Inst. for Biological Studies, La Jolla, CA (United States))

    1992-02-15

    A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCr analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

  15. Major West Indies MRSA clones in human beings: do they travel with their hosts?

    Science.gov (United States)

    Chroboczek, Tomasz; Boisset, Sandrine; Rasigade, Jean-Philippe; Meugnier, Helene; Akpaka, Patrick E; Nicholson, Alison; Nicolas, Muriel; Olive, Claude; Bes, Michele; Vandenesch, François; Laurent, Frederic; Etienne, Jerome; Tristan, Anne

    2013-01-01

    Descriptions of the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) have seldom been produced in the Caribbean, which is a major tourism destination. Using DNA microarrays and spa typing, we characterized 85 MRSA isolates from human skin and soft-tissue infections from five different islands. In the French West Indies (n = 72), the most frequently isolated clones were the same clones that are specifically isolated from mainland France [Lyon (n = 35) and Geraldine (n = 11) clones], whereas the clones that were most frequently isolated from the other islands (n = 13) corresponded with clones that have a worldwide endemic spread [Vienna/Hungarian/Brazilian (n = 5), Panton Valentine leukocidin-positive USA300 (n = 4), New York/Japan (n = 2), and pediatric (n = 1) clones]. The distribution of the major MRSA clones in the French (Guadeloupe and Martinique) and non-French West Indies (Jamaica, Trinidad, and Tobago) is different, and the clones most closely resemble those found in the home countries of the travelers who visit the islands most frequently. The distribution might be affected by tourist migration, which is specific to each island. © 2013 International Society of Travel Medicine.

  16. A resource-based version of the argument that cloning is an affront to human dignity.

    Science.gov (United States)

    McDougall, R

    2008-04-01

    The claim that human reproductive cloning constitutes an affront to human dignity became a familiar one in 1997 as policymakers and bioethicists responded to the announcement of the birth of Dolly the sheep. Various versions of the argument that reproductive cloning is an affront to human dignity have been made, most focusing on the dignity of the child produced by cloning. However, these arguments tend to be unpersuasive and strongly criticised in the bioethical literature. In this paper I put forward a different argument that reproductive cloning is an affront to human dignity, one that looks beyond the dignity of the child produced. I suggest that allocating funds to such a pursuit can affront human dignity by diverting resources away from those existing people who lack sufficient health to enable them to exercise basic rights and liberties. This version of the argument posits cloning as an affront to human dignity in particular circumstances, rather than claiming the technology as intrinsically inconsistent with human dignity.

  17. Identification of putative human T cell receptor delta complementary DNA clones

    Energy Technology Data Exchange (ETDEWEB)

    Hata, S.; Brenner, M.B.; Krangel, M.S.

    1987-10-30

    A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCY ..gamma.. and CD3 polypeptides, were recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR..gamma..delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR ..gamma..delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR ..gamma..delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics are strong evidence that the complementary DNA clones encode TCR delta.

  18. Human growth hormone binding and stimulation of insulin biosynthesis in cloned rat insulinoma cells

    DEFF Research Database (Denmark)

    Billestrup, Nils

    1985-01-01

    Binding of 125I labelled human growth hormone to cloned insulin producing RIN-5AH cells is described. Binding was specific for somatotropic hormones since both human and rat growth hormone could compete for binding sites, whereas much higher concentrations of lactogenic hormones were needed...

  19. Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    JI Jingjuan; GUO Tonghang; TONG Xianhong; LUO Lihua; ZHOU Guixiang; FU Yingyun; LIU Yusheng

    2007-01-01

    Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P<0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.

  20. Human cloning and embryo research: the 2003 John J. Conley Lecture on medical ethics.

    Science.gov (United States)

    George, Robert P

    2004-01-01

    The author, a member of the U.S. President's Council on Bioethics, discusses ethical issues raised by human cloning, whether for purposes of bringing babies to birth or for research purposes. He first argues that every cloned human embryo is a new, distinct, and enduring organism, belonging to the species Homo sapiens, and directing its own development toward maturity. He then distinguishes between two types of capacities belonging to individual organisms belonging to this species, an immediately exerciseable capacity and a basic natural capacity that develops over time. He argues that it is the second type of capacity that is the ground for full moral respect, and that this capacity (and its concomitant degree of respect) belongs to cloned human embryos no less than to adult human beings. He then considers and rejects counter-arguments to his position, including the suggestion that the capacity of embryos is equivalent to the capacity of somatic cells, that full human rights are afforded only to human organisms with functioning brains, that the possibility of twinning diminishes the moral status of embryos, that the fact that people do not typically mourn the loss of early embryos implies that they have a diminished moral status, that the fact that early spontaneous abortions occur frequently diminishes the moral status of embryos, and that his arguments depend upon a concept of ensoulment. He concludes that if the moral status of cloned human embryos is equivalent to that of adults, then public policy should be based upon this assumption.

  1. Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline.

    Science.gov (United States)

    Da Cunha, Violette; Davies, Mark R; Douarre, Pierre-Emmanuel; Rosinski-Chupin, Isabelle; Margarit, Immaculada; Spinali, Sebastien; Perkins, Tim; Lechat, Pierre; Dmytruk, Nicolas; Sauvage, Elisabeth; Ma, Laurence; Romi, Benedetta; Tichit, Magali; Lopez-Sanchez, Maria-José; Descorps-Declere, Stéphane; Souche, Erika; Buchrieser, Carmen; Trieu-Cuot, Patrick; Moszer, Ivan; Clermont, Dominique; Maione, Domenico; Bouchier, Christiane; McMillan, David J; Parkhill, Julian; Telford, John L; Dougan, Gordan; Walker, Mark J; Holden, Matthew T G; Poyart, Claire; Glaser, Philippe

    2014-08-04

    Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

  2. Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction.

    Science.gov (United States)

    Songsivilai, S; Bye, J M; Marks, J D; Hughes-Jones, N C

    1990-12-01

    Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.

  3. Tetranectin, a plasminogen kringle 4-binding protein. Cloning and gene expression pattern in human colon cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Albrechtsen, R

    1992-01-01

    BACKGROUND: Tetranectin is a recently discovered protein that binds to kringle 4 region of plasminogen (Clemmensen I, Petersen LC, Kluft C. Eur J Biochem 1986; 156:327. EXPERIMENTAL DESIGN: The mRNA encoding human tetranectin was cloned by using degenerate primers in a reverse transcriptase...... reaction followed by polymerase chain reaction amplification. The resulting polymerase chain reaction product was examined by DNA sequencing and subsequently used as probe for screening a human placental cDNA library. A full length cDNA clone (TET-1) was isolated, characterized, and used for Northern blot...

  4. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  5. Dengue virus-specific, human CD4+ CD8- cytotoxic T-cell clones: multiple patterns of virus cross-reactivity recognized by NS3-specific T-cell clones.

    OpenAIRE

    Kurane, I; Brinton, M.A.; Samson, A L; Ennis, F A

    1991-01-01

    Thirteen dengue virus-specific, cytotoxic CD4+ CD8- T-cell clones were established from a donor who was infected with dengue virus type 3. These clones were examined for virus specificity and human leukocyte antigen (HLA) restriction in cytotoxic assays. Six patterns of virus specificities were determined. Two serotype-specific clones recognized only dengue virus type 3. Two dengue virus subcomplex-specific clones recognized dengue virus types 2, 3, and 4, and one subcomplex-specific clone re...

  6. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  7. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  8. Prosthetic clone and natural human tooth comparison by speckle interferometry

    Science.gov (United States)

    Slangen, Pierre; Corn, Stephane; Fages, Michel; Raynal, Jacques; Cuisinier, Frederic J. G.

    2010-09-01

    New trends in dental prosthodontic interventions tend to preserve the maximum of "body" structure. With the evolution of CAD-CAM techniques, it is now possible to measure "in mouth" the remaining dental tissues. The prosthetic crown is then designed using this shape on which it will be glued on, and also by taking into account the contact surface of the opposite jaw tooth. Several theories discuss on the glue thickness and formulation, but also on the way to evolve to a more biocompatible crown and also new biomechanical concepts. In order to validate these new concepts and materials, and to study the mechanical properties and mechanical integrity of the prosthesis, high resolution optical measurements of the deformations of the glue and the crown are needed. Samples are two intact premolars extracted for orthodontics reasons. The reference sample has no modifications on the tooth while the second sample tooth is shaped to receive a feldspathic ceramic monoblock crown which will be glued. This crown was manufactured with a chairside CAD-CAM system from an intra-oral optical print. The software allows to realize a nearly perfect clone of the reference sample. The necessary space for the glue is also entered with ideal values. This duplication process yields to obtain two samples with identical anatomy for further processing. The glue joint thickness can also be modified if required. The purpose is to compare the behaviour of a natural tooth and its prosthetic clone manufactured with "biomechanical" concepts. Vertical cut samples have been used to deal with planar object observation, and also to look "inside" the tooth. We have developed a complete apparatus enabling the study of the compressive mechanical behaviour of the concerned tooth by speckle interferometry. Because in plane displacements are of great interest for orthodontic measurements1, an optical fiber in-plane sensitive interferometer has been designed. The fibers are wrapped around piezoelectric

  9. Th1-like human T-cell clones recognizing Leishmania gp63 inhibit Leishmania major in human macrophages

    DEFF Research Database (Denmark)

    Kemp, M; Hey, A S; Bendtzen, K

    1994-01-01

    The major surface protease of Leishmania major, gp63, has been suggested as a vaccine candidate for cutaneous leishmaniasis. In this study gp63 was purified from L. major promastigotes. A panel of human T-cell clones recognizing this protein were generated from individuals who had previously had...... self-healing cutaneous leishmaniasis. The T-cell clones expressed CD4, and the alpha chain of the T-cell antigen receptor. GP63 reactive T-cell clones activated by antigen or by immobilized anti-CD3 antibody released relative large amounts of interferon-gamma and no or little interleukin-4, thereby...... resembling Th1 cells. Autologous mononuclear cells and Epstein-Barr virus-transformed B cell lines were equally efficient in presenting the antigen to the T cells. The gp63 reactive T cells induced resistance to infection in cultured human macrophages by L. major. The data confirm that human CD4+ T cells...

  10. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S;

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...

  11. Integration of the cytogenetic, genetic, and physical maps of the human genome by FISH mapping of CEPH YAC clones

    Energy Technology Data Exchange (ETDEWEB)

    Bray-Ward, P.; Menninger, J.; Lieman, J. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others

    1996-02-15

    This article discusses the genetic mapping of over 950 yeast artificial chromosome (YAC) clones on human chromosomes. This integration of the cytogenetic, genetic and physical maps of the human genome was accomplished using fluorescence in situ hybridization (FISH) mapping and the CEPH library of YAC clones. 27 refs., 2 figs., 1 tab.

  12. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were us...

  13. Cloning, characterization, and mRNA expression analysis of novel human fetal cochlear cDNAs.

    NARCIS (Netherlands)

    Luijendijk, M.W.J.; Pol, T.J.R. van de; Duijnhoven, G.C.F. van; Hollander, A.I. den; Caat, J. ten; Limpt, V. van; Brunner, H.G.; Kremer, J.M.J.; Cremers, F.P.M.

    2003-01-01

    To identify novel genes that are expressed specifically or preferentially in the cochlea, we constructed a cDNA library enriched for human cochlear cDNAs using a suppression subtractive hybridization technique. We analyzed 2640 clones by sequencing and BLAST similarity searches. One hundred and fift

  14. Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

    DEFF Research Database (Denmark)

    Cowell, G M; Kønigshøfer, E; Danielsen, E M

    1988-01-01

    The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

  15. Can artificial parthenogenesis sidestep ethical pitfalls in human therapeutic cloning? An historical perspective

    Science.gov (United States)

    Fangerau, H

    2005-01-01

    The aim of regenerative medicine is to reconstruct tissue that has been lost or pathologically altered. Therapeutic cloning seems to offer a method of achieving this aim; however, the ethical debate surrounding human therapeutic cloning is highly controversial. Artificial parthenogenesis—obtaining embryos from unfertilised eggs—seems to offer a way to sidestep these ethical pitfalls. Jacques Loeb (1859–1924), the founding father of artificial parthogenesis, faced negative public opinion when he published his research in 1899. His research, the public's response to his findings, and his ethical foundations serve as an historical argument both for the communication of science and compromise in biological research. PMID:16319240

  16. The search for truth and freedom: ethical issues surrounding human cloning and stem cell research.

    Science.gov (United States)

    Bruce, Alex

    2002-02-01

    The reality of cloning and stem cell research has provoked wonder, fear and anger. These developments have the potential fundamentally to alter humanity. But how well informed is the range of views being expressed? Is progress being threatened by understandable but uninformed fears? Or are scientists rushing toward an ethical abyss, so concerned with what they can do that they never stop to ask what they should do? This article identifies some of the fears and hopes surrounding cloning and stem cell research. It aims to provoke ethical debate in evaluating such research.

  17. Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

    Directory of Open Access Journals (Sweden)

    Kalpana Dulal

    2012-01-01

    Full Text Available Bacterial artificial chromosome (BAC technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV. The HCMV BAC clone propagated and maintained inside E. coli allows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method in E. coli for molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects for in vitro homologous recombination for genetic cloning.

  18. [Structural organization of the human p53 gene. I. Molecular cloning of the human p53 gene].

    Science.gov (United States)

    Bukhman, V L; Ninkina, N N; Chumakov, P M; Khilenkova, M A; Samarina, O P

    1987-09-01

    Human p53 gene was cloned from the normal human placenta DNA and DNA from the strain of human kidney carcinoma transplanted into nude mice. Representative gene library from tumor strain of human kidney carcinoma and library of 15 kb EcoRI fragments of DNA from normal human placenta were constructed. Maniatis gene library was also used. Five clones were isolated from kidney carcinoma library; they covered 27 kb and included full-length p53 gene of 19.5 kb and flanking sequences. From normal placenta libraries three overlapped clones were obtained. Restriction map of cloned sequences was constructed and polarity of the p53 gene determined. The first intron of the gene is large (10.4 kb); polymorphic BglII site was observed in this intron, which allows to discriminate between allelic genes. One of these (BglII-) is ten times more abundant that the other (BglII+). Both allelic genes are able to synthesize the 2.8 kb p53 gene.

  19. Statistical analysis of clone formation in cultures of human stem cells.

    Science.gov (United States)

    Bochkov, N P; Vinogradova, M S; Volkov, I K; Voronina, E S; Kuleshov, N P

    2011-08-01

    We performed a statistical analysis of clone formation from aneuploid cells (chromosomes 6, 8, 11, X) in cultures of bone marrow-derived human multipotent mesenchymal stromal cells by spontaneous level of aneuploidy at different terms of culturing (from 2 to 19 cell cycles). It was found that the duration of cell cycle increased from 65.6 h at passages 2-3 to 164.5 h at passage 12. The expected ratio of aneuploid cells was calculated using modeled 5, 10, 20 and 30% selective preference in reproduction. The size of samples for detecting 10, 25, and 50% increased level of aneuploidy was calculated. The presented principles for evaluation of aneuploid clone formation may be used to distinguish clones of any abnormal cells.

  20. Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

    Energy Technology Data Exchange (ETDEWEB)

    Antonarakis, S.E.

    1991-09-01

    The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

  1. Prohibited anabolic substances, dangerous to human consumers, in accordance of E.U. regulations

    Directory of Open Access Journals (Sweden)

    Flavia A. Hanganu,

    2009-06-01

    Full Text Available Substances with anabolic effects are used to enhance feed conversion, growth rate or muscle tissue deposition in stock farming for several decades. The majority of these compounds have biochemical effects similar to sex steroids (androgens, estrogens, gestagens. However, in the E.U., the use of hormones for growth – promotion or fattening is prohibited. Monitoring of residues of hormonal growth-promotion in meat or milk, is essential for implementing such bans and to protect public health against the harmful effects of these substances, which incidentally is found in products of animal origin.

  2. Human body motion tracking based on quantum-inspired immune cloning algorithm

    Science.gov (United States)

    Han, Hong; Yue, Lichuan; Jiao, Licheng; Wu, Xing

    2009-10-01

    In a static monocular camera system, to gain a perfect 3D human body posture is a great challenge for Computer Vision technology now. This paper presented human postures recognition from video sequences using the Quantum-Inspired Immune Cloning Algorithm (QICA). The algorithm included three parts. Firstly, prior knowledge of human beings was used, the key joint points of human could be detected automatically from the human contours and skeletons which could be thinning from the contours; And due to the complexity of human movement, a forecasting mechanism of occlusion joint points was addressed to get optimum 2D key joint points of human body; And then pose estimation recovered by optimizing between the 2D projection of 3D human key joint points and 2D detection key joint points using QICA, which recovered the movement of human body perfectly, because this algorithm could acquire not only the global optimal solution, but the local optimal solution.

  3. Sympathy for the Clone: (PostHuman Identities Enhanced by the ‘Evil Science’ Construct and its Commodifying Practices in Contemporary Clone Fiction

    Directory of Open Access Journals (Sweden)

    Jimena Escudero Pérez

    2014-12-01

    Full Text Available The manipulation of human DNA in the form of eugenic pursuit, cloning, genetic engineering etc., has become a well-established subject in science fiction for decades now. In our days, this thematic trend is probably the most prolific one when inspiring narratives in popular culture and also constitutes the source of much bioethical debate. A common pattern derived from these practices is that they generate dystopian scenarios where a community is oppressed and abused by scientific means thus portraying science and its agents as evil. In the case of clone fiction, the focus of this article, the inhumanity of the oppressive powers enhances the questioned humanity of the clones, a particularly complex and evolving type of character that is often commodified. This paper analyses the "evil science" construction and the semiotics of the human/clone identity it produces as displayed in the cases of Never Let Me Go (Kazuo Ishiguro, 2005, The Island (Dir. Michael Bay, USA, 2005, Moon (Dir. Duncan Jones, UK, 2009 and the TV series Orphan Black (created by Graeme Manson & John Fawcett, Canada, 2013–. The cited examples provide references for typified patterns as well as for the development of both the clone figure and the scientific evilness component.

  4. Livestock Origin for a Human Pandemic Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus

    DEFF Research Database (Denmark)

    Spoor, Laura E.; McAdam, Paul R.; Weinert, Lucy A.;

    2013-01-01

    ABSTRACT The importance of livestock as a source of bacterial pathogens with the potential for epidemic spread in human populations is unclear. In recent years, there has been a global increase in community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections of healthy...... of emergent clones of methicillin-resistant Staphylococcus aureus (MRSA) that originated in livestock and switched to humans, followed by host-adaptive evolution and epidemic spread in global human populations. Our findings demonstrate that livestock can act as a reservoir for the emergence of new human...

  5. Cloning, chromosome localization and features of a novel human gene, MATH2

    Indian Academy of Sciences (India)

    Lingchen Guo; Min Jiang; Yushu Ma; Haipeng Cheng; Xiaohua Ni; Yangsheng Jin; Yi Xie; Yumin Mao

    2002-04-01

    We report cloning and some features of a novel human gene, MATH2, which encodes a protein of 337 amino acid residues with a basic helix–loop–helix domain and exhibits 98% similarity to mouse Math2. Results of Northern blot analysis revealed two transcripts of the MATH2 gene of 1.7 kb and 2.4 kb in human brain. We localized MATH2 to chromosome 7 at 7p14–15 by matching with the Human Genome Sequence Database. Human MATH2 and mouse Math2 may have the same functions in the nervous system.

  6. [Triploid cloned human embryos: ethical, social, and legal aspects].

    Science.gov (United States)

    Bellver Capella, Vicente

    2012-01-01

    This work attempts to place the experiment within the scientific and social framework of pluripotent-stem-cell research and offer reflections of an ethical and (to a lesser extent) legal nature on the results obtained by this research group. To these ends, the work is divided into two parts. The first part describes the most important aspects of Noggle and Egli's announcement and the biotechnological and media context in which it was made. The second part is concerned with the bioethical issues raised by the experiment. There are basically four issues, which relate to: (1) the nuclear transfer technique, (2) the use of human ovules to carry out the experiment, (3) the destruction of human blastocysts, and (4) the ethical requirements of scientific publications.

  7. Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

    Science.gov (United States)

    Bautsch, W; Emde, M; Kretzschmar, T; Köhl, J; Suckau, D; Bitter-Suermann, D

    1992-06-01

    A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

  8. Human nicotinamide N-methyltransferase gene: Molecular cloning, structural characterization and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Aksoy, S.; Weinshilboum, R.M. [Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, MN (United States); Brandriff, B.F. [Lawrence Livermore National Lab., CA (United States); Ward, A.; Little, P.F.R. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

    1995-10-10

    Genomic DNA clones for nicotinamide N-methyltransferase (NNMT), an enzyme that catalyzes drug and xenobiotic metabolism, were isolated from a human chromosome 11-specific DNA library. Study of one of those clones, when combined with PCR-based experiments performed with human genomic DNA, made it possible to determine the structure of the human NNMT gene. The gene was approximately 16.5 kb in length and consisted of 3 exons and 2 introns. Transcription initiation for the NNMT gene occurred 105-109 nucleotides 5{prime}-upstream from the cDNA translation initiation codon on the basis of the results of both primer extension and 5{prime}-rapid amplification of cDNA ends. NNMT mapped to chromosome band 11q23.1 by fluorescence in situ hybridization.

  9. Nucleotide sequence of cloned cDNA for human pancreatic kallikrein.

    Science.gov (United States)

    Fukushima, D; Kitamura, N; Nakanishi, S

    1985-12-31

    Cloned cDNA sequences for human pancreatic kallikrein have been isolated and determined by molecular cloning and sequence analysis. The identity between human pancreatic and urinary kallikreins is indicated by the complete coincidence between the amino acid sequence deduced from the cloned cDNA sequence and that reported partially for urinary kallikrein. The active enzyme form of the human pancreatic kallikrein consists of 238 amino acids and is preceded by a signal peptide and a profragment of 24 amino acids. A sequence comparison of this with other mammalian kallikreins indicates that key amino acid residues required for both serine protease activity and kallikrein-like cleavage specificity are retained in the human sequence, and residues corresponding to some external loops of the kallikrein diverge from other kallikreins. Analyses by RNA blot hybridization, primer extension, and S1 nuclease mapping indicate that the pancreatic kallikrein mRNA is also expressed in the kidney and sublingual gland, suggesting the active synthesis of urinary kallikrein in these tissues. Furthermore, the tissue-specific regulation of the expression of the members of the human kallikrein gene family has been discussed.

  10. Evaluation of cloned cells, animal model, and ATRA sensitivity of human testicular yolk sac tumor

    Directory of Open Access Journals (Sweden)

    Zhao Junfeng

    2012-03-01

    Full Text Available Abstract The testicular yolk sac tumor (TYST is the most common neoplasm originated from germ cells differentiated abnormally, a major part of pediatric malignant testicular tumors. The present study aimed at developing and validating the in vitro and vivo models of TYST and evaluating the sensitivity of TYST to treatments, by cloning human TYST cells and investigating the histology, ultra-structure, growth kinetics and expression of specific proteins of cloned cells. We found biological characteristics of cloned TYST cells were similar to the yolk sac tumor and differentiated from the columnar to glandular-like or goblet cells-like cells. Chromosomes for tumor identification in each passage met nature of the primary tumor. TYST cells were more sensitive to all-trans-retinoic acid which had significantly inhibitory effects on cell proliferation. Cisplatin induced apoptosis of TYST cells through the activation of p53 expression and down-regulation of Bcl- expression. Thus, we believe that cloned TYST cells and the animal model developed here are useful to understand the molecular mechanism of TYST cells and develop potential therapies for human TYST.

  11. NotI linking clones as a tool for joining physical and genetic maps of the human genome.

    Science.gov (United States)

    Allikmets, R L; Kashuba, V I; Pettersson, B; Gizatullin, R; Lebedeva, T; Kholodnyuk, I D; Bannikov, V M; Petrov, N; Zakharyev, V M; Winberg, G

    1994-01-15

    To study the connection among NotI linking clones, CpG islands, and genes, the sequence surrounding 143 NotI sites was determined. These NotI linking clones were isolated from human chromosome 3-specific libraries and contain an average C + G content of 65%. These clones represent sequence-tagged sites that can be positioned onto chromosome maps and used for generating a long-range NotI map of the human genome. A majority (about 90%) of these clones contain transcribed sequences, as detected by Northern blot hybridization, providing an efficient link between physical and functional (genetic) maps. The GenBank nucleotide database was searched with sequences from these NotI linking clones. For many clones, homology was found to human and other vertebrate genes. About 20 clones contained various repeats in their sequences and may represent microsatellite loci. Most of these NotI linking clones therefore represent evolutionarily conserved DNA fragments and also can be used for comparative genome mapping of other mammalian species. In addition, approximately 20% of all sequenced human CpG island-containing genes and more than 12% of all well-characterized human genes were found to possess NotI restriction sites. This is at least 2-5 times more than has been previously estimated and suggests that NotI sites have a much stronger association with genes.

  12. NotL linking clones as a tool for joining physical and genetic maps of the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Allikmets, R.L.; Dean, M.; Modi, W. (DynCorp National Cancer Institute, Frederick, MD (United States)); Kholodnyuk, I.D.; Winberg, G.; Klein, G. (Karolinska Institutet, Stockholm (Sweden)); Pettersson, B.; Uhlen, M. (Royal Institute of Technology, Stockholm (Sweden)); Gizatullin, R.; Bannikov, V.M. (and others)

    1994-01-15

    To study the connection among NotI linking clones, CpG islands, and genes, the sequence surrounding 143 NotI sites was determined. These NotI linking clones were isolated from human chromosome 3-specific libraries and contain an average C + G content of 65%. These clones represent sequence-tagged sites that can be positioned onto chromosome maps and used for generating a long-range NotI map of the human genome. A majority (about 90%) of these clones contain transcribed sequences, as detected by Northern blot hybridization, providing an efficient link between physical and functional (genetic) maps. The GenBank nucleotide database was searched with sequences from these NotI linking clones. For many clones, homology was found to human and other vertebrate genes. About 20 clones contained various repeats in their sequences and may represent microsatellite loci. Most of these NotI linking clones therefore represent evolutionarily conserved DNA fragments and also can be used for comparative genome mapping of other mammalian species. In addition, approximately 20% of all sequenced human CpG island-containing genes and more than 12% of all well-characterized human genes were found to possess NotI restriction sites. This is at least 2-5 times more than has been previously estimated and suggests that NotI sites have a much stronger association with genes. 41 refs., 3 figs., 2 tabs.

  13. What justifies the United States ban on federal funding for nonreproductive cloning?

    Science.gov (United States)

    Cunningham, Thomas V

    2013-11-01

    This paper explores how current United States policies for funding nonreproductive cloning are justified and argues against that justification. I show that a common conceptual framework underlies the national prohibition on the use of public funds for cloning research, which I call the simple argument. This argument rests on two premises: that research harming human embryos is unethical and that embryos produced via fertilization are identical to those produced via cloning. In response to the simple argument, I challenge the latter premise. I demonstrate there are important ontological differences between human embryos (produced via fertilization) and clone embryos (produced via cloning). After considering the implications my argument has for the morality of publicly funding cloning for potential therapeutic purposes and potential responses to my position, I conclude that such funding is not only ethically permissible, but also humane national policy.

  14. Molecular cloning and structural analysis of human norepinephrine transporter gene(NETHG)

    Institute of Scientific and Technical Information of China (English)

    GUOLIHE; LIHUAZHU; 等

    1995-01-01

    A cDNA molecule encoding a major part of the human Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene.A positive clone harbouring the whole gene was obtained from a human lymphocyte genomic library through utilizing the “genomic walking” technique.The clone,designated as phNET,harbours a DNA fragment of about 59 kd in length inserted into BamH I site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein.A single exon encodes a distinct transmembrane domain,except for transmembrane domain 10 and 11,which are encoded by part of two exons respectively,and exon 12,which encodes part of domain 11 and all of domain 12.These results imply that there is a close relationship between exon splicing of a gene and structureal domains of the protein,as is the case for the human γ-aminobutyric acid transporter(hGAT) and a number of other membrane proteins.

  15. Development of High-Throughput Phenotyping of Metagenomic Clones from the Human Gut Microbiome for Modulation of Eukaryotic Cell Growth▿

    OpenAIRE

    2007-01-01

    Metagenomic libraries derived from human intestinal microbiota (20,725 clones) were screened for epithelial cell growth modulation. Modulatory clones belonging to the four phyla represented among the metagenomic libraries were identified (hit rate, 0.04 to 8.7% depending on the screening cutoff). Several candidate loci were identified by transposon mutagenesis and subcloning.

  16. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor (interleukin 5)

    Energy Technology Data Exchange (ETDEWEB)

    Campbell, H.D.; Tucker, W.Q.J.; Hort, Y.; Martinson, M.E.; Mayo, G.; Clutterbuck, E.J.; Sanderson, C.J.; Young, I.G.

    1987-10-01

    The human eosinophil differentiation factor (EDF) gene was cloned from a genomic library in lambda phage EMBL3A by using a murine EDF cDNA clone as a probe. The DNA sequence of a 3.2-kilobase BamHI fragment spanning the gene was determined. The gene contains three introns. The predicted amino acid sequence of 134 amino acids is identical with that recently reported for human interleukin 5 but shows no significant homology with other known hemopoietic growth regulators. The amino acid sequence shows strong homology (approx. 70% identity) with that of murine EDF. Recombinant human EDF, expressed from the human EDF gene after transfection into monkey COS cells, stimulated the production of eosinophils and eosinophil colonies from normal human bone marrow but had no effect on the production of neutrophils or mononuclear cells (monocytes and lymphoid cells). The apparent specificity of human EDF for the eosinophil lineage in myeloid hemopoiesis contrasts with the properties of human interleukin 3 and granulocyte/macrophage and granulocyte colony-stimulating factors but is directly analogous to the biological properties of murine EDF. Human EDF therefore represents a distinct hemopoietic growth factor that could play a central role in the regulation of eosinophilia.

  17. Rabbit muscle creatine phosphokinase. CDNA cloning, primary structure and detection of human homologues.

    Science.gov (United States)

    Putney, S; Herlihy, W; Royal, N; Pang, H; Aposhian, H V; Pickering, L; Belagaje, R; Biemann, K; Page, D; Kuby, S

    1984-12-10

    A cDNA library was constructed from rabbit muscle poly(A) RNA. Limited amino acid sequence information was obtained on rabbit muscle creatine phosphokinase and this was the basis for design and synthesis of two oligonucleotide probes complementary to a creatine kinase cDNA sequence which encodes a pentapeptide. Colony hybridizations with the probes and subsequent steps led to isolation of two clones, whose cDNA segments partially overlap and which together encode the entire protein. The primary structure was established from the sequence of two cDNA clones and from independently determined sequences of scattered portions of the polypeptide. The reactive cysteine has been located to position 282 within the 380 amino acid polypeptide. The rabbit cDNA hybridizes to digests of human chromosomal DNA. This reveals a restriction fragment length polymorphism associated with the human homologue(s) which hybridizes to the rabbit cDNA.

  18. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  19. United Nations and human cloning: a slender and fortunate defence for biomedical research.

    Science.gov (United States)

    Edwards, R G

    2003-12-01

    Numerous biomedical scientists have contributed to the wide knowledge on the growth of preimplantation human embryos in vitro, now improving every aspect of the form of clinical care. These data were gained ethically in many countries, to open new vistas including the alleviation of infertility, preimplantation genetic diagnosis and stem cells, combined with some recent reports on human reproductive cloning. After detailed consultations with scientists, clinicians, ethicists and lawyers, many governments passed legislation permitting research under their own particular socially-defined conditions. Virtually all of them rejected reproductive cloning; a few have accepted therapeutic cloning. These legislatures saluted the many biomedical scientists striving to improve IVF and its derivatives, recognizing their immense medical potential. A motion recently placed before the United Nations then recommended a worldwide ban on all forms of human cloning. Proponents included the Vatican and many Roman Catholic countries, the USA and others. Opponents included Belgium, China, Japan, Brazil, UK, Germany and France. Mediation was achieved by Iran and other Muslim nations, and led to a motion passed by single vote for a two-year delay. This may be the first-ever proposal to ban worldwide a particular form of research. It sounds the alarm bells for further research. It raises questions about the UN being an appropriate forum for ethical decisions affecting the entire world and its future medicine. Large blocs of nations committed to particular religions and outlooks confronted each other, a situation in total contrast to the detailed and widespread consultations made by individual governments when deciding their own individual ethics. This event was clearly a narrow escape for free research as defined by each country's own jurisprudence. It also places research on human embryology and reproductive biomedicine into a more critical situation than before. Current liberalism in

  20. Cloned human neuropeptide Y receptor couples to two different second messenger systems.

    OpenAIRE

    Herzog, H.; Hort, Y J; Ball, H J; Hayes, G; Shine, J; Selbie, L A

    1992-01-01

    Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmacological criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY rece...

  1. Rights and wrongs under the ECHR : The prohibition of abuse of rights in Article 17 of the European Convention on Human Rights

    NARCIS (Netherlands)

    de Morree, P.E.

    2016-01-01

    The prohibition of abuse of rights in Article 17 of the European Convention on Human Rights (ECHR or Convention) embodies one of the Convention’s main principles: its commitment to democracy and democratic values. The provision aims to prevent groups and individuals from successfully invoking fundam

  2. Cloning and Identification of A Novel Variant of Human Vascular Endothelial Growth Factor

    Institute of Scientific and Technical Information of China (English)

    GUO; Jianli; QU; Shen

    2001-01-01

    A novel variant of human vascular endothelial growth factor (h'VEGF165) cDNA was amplified by nested PCR method from the HL601 cells and was cloned into a eukaryotic expressing vector pcDNA3 to construct a recombinant plasmid pCD-h'VEGF165. The amplified h'VEGF165cDNA fragment was identified by enzyme digestion and DNA sequencing methods. Also, wild-type hVEGF165 cDNA was obtained, identified and cloned into a eukaryotic expressing vector pcDNA3by using the same methods. The results of DNA sequencing showed that h'VEGF165 cDNA cloned from HL601 was 600 bp in size with 8 % of the base sequence in h'VEGF165 cDNA being changed as compared with the base sequence in the wild-type hVEGF165 cDNA. The results of sequencing of hVEGF165 which was cloned from HL60 by us were consistent with the reports completely.

  3. Ethical consideration of experimentation using living human embryos: the Catholic Church's position on human embryonic stem cell research and human cloning.

    Science.gov (United States)

    Ohara, N

    2003-01-01

    Although the potential applications of human embryonic stem cells and therapeutic cloning hold promise for the alleged medical benefits, these technologies have posed profound ethical issues because they necessitate the destruction of human embryos. A fundamental point in the issues is the concept of the moral status of human embryos. The Catholic Church has held that human life begins at the moment of conception and therefore, has defended the dignity, inviolable right to life and integrity of human embryos. The Catholic Church has opposed human embryonic stem cell research and any kind of human cloning because they are contrary to the dignity of procreation, of conjugal union and of human embryos. Moreover, these techniques have the risk of creating a sub-category of human beings that are destined basically for the convenience of others. In conclusion, science and technology can never be independent of the criterion of morality, since technology exists for man and must respect his finality.

  4. Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry

    Energy Technology Data Exchange (ETDEWEB)

    Aldred, P.; Fu, Ping; Barrett, G.; Penschow, J.D.; Wright, R.D.; Coghlan, J.P.; Fernley, R.T. (The Howard Florey Institute of Experimental Physiology and Medicine, Parkville, Victoria (Australia))

    1991-01-01

    Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CAVI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension. Overall the human CA VI protein has a sequence identity of 35 {percent} with human CA II, while residues involved in the active site of the enzymes have been conserved. The human and sheep secreted carbonic anhydrases have a sequence identity of 72 {percent}. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

  5. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  6. Cloning and expression of a novel human HCUTA cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Copper is one of the most important trace elements to life. Human HCUTA is a novel cDNA encoding a 156aa protein, which may participate in human copper tolerance system. The HCUTA protein is highly similar to protein CUT A1 of E. coli. The whole opening reading frame of HCUTA cDNA was amplified by PCR and cloned into pET28a + express vector, and the HCUTA protein was effectively expressed in E. coli BL21 (DE3).

  7. The Singapore approach to human stem cell research, therapeutic and reproductive cloning.

    Science.gov (United States)

    Kian, Catherine Tay Swee; Leng, Tien Sim

    2005-06-01

    With the controversial ethical issues on the creation of human embryos through cloning for therapeutic research, which holds more promise of medical breakthroughs that the world could ever imagine and the acknowledgement by many scientists that this technology may not lead in the near future to therapies; this country report discusses the approach Singapore takes on human stem cell research, interjected with the authors' own arguments and suggestions especially on research compensation injuries, an often neglected important issue. International comparative viewpoints taken by the major countries in the world are also included in the appendix.

  8. Using therapeutic cloning to fight human disease: a conundrum or reality?

    Science.gov (United States)

    Hall, Vanessa J; Stojkovic, Petra; Stojkovic, Miodrag

    2006-07-01

    The development and transplantation of autologous cells derived from nuclear transfer embryonic stem cell (NT-ESC) lines to treat patients suffering from disease has been termed therapeutic cloning. Human NT is still a developing field, with further research required to improve somatic cell NT and human embryonic stem cell differentiation to deliver safe and effective cell replacement therapies. Furthermore, the implications of transferring mitochondrial heteroplasmic cells, which may harbor aberrant epigenetic gene expression profiles, are of concern. The production of human NT-ESC lines also remains plagued by ethical dilemmas, societal concerns, and controversies. Recently, a number of alternate therapeutic strategies have been proposed to circumvent the moral implications surrounding human nuclear transfer. It will be critical to overcome these biological, legislative, and moral restraints to maximize the potential of this therapeutic strategy and to alleviate human disease.

  9. Cloning the mouse homologue of the human lysosomal acid {alpha}-glucosidase gene

    Energy Technology Data Exchange (ETDEWEB)

    Ding, J.H.; Yang, B.Z.; Liu, H.M. [Duke Univ. Medical Center, Durham, NC (United States)] [and others

    1994-09-01

    Pompe disease (GSD II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid {alpha}-glucosidase (GAA). In an attempt to create a mouse model for Pompe disease, we isolated and characterized the gene encoding the mouse homologue of the human GAA. Twenty clones that extend from exon 2 to the poly(A) tail were isolated from a mouse liver cDNA library, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-2 were cloned by RACE from mouse liver RNA. The full-length liver GAA cDNA contains 3365 nucleotides with a coding region of 2859 nucleotides and a 394 base pair 3{prime}-nontranslated region. The deduced amino acid sequence of the mouse GAA shows 84% identity to the human GAA. Southern blot analysis demonstrated that the mouse GAA was encoded by a single copy gene. Then six bacteriophages containing DNA from the GAA gene were isolated by screening 10{sup 6} phage plaques of a mouse 129 genomic library using a mouse GAA cDNA as a probe. From one of these bacteriophages, an 11-kilobase EcoRI fragment containing exons 3 to 15 was subcloned and sequenced. Work is in progress using this genomic clone to disrupt the GAA gene in murine embryonic stem cells in order to create GSD II mice.

  10. Heterogeneity of CD4-Positive Human T-Cell Clones Which Recognize the Surface Protein Antigen of Rickettsia typhi

    Science.gov (United States)

    1989-04-01

    5055 " - PROGRAM PROJECT 1TASK IWORK UNIT ELEMENT NO. NO. / NO. IACC SSI II. TITLE (Incluae Security Clasification ) Heterogeneity o. CD4-Positive...Human T-Cell Clones Which Kecognize the Surface Protein Antigen of Rickettsia typhi 12. PERSONAL AOTU-OR(S) Carl M, Vaidya S, Robbins FM, Ching WM...Heterogeneity of CD4-Positive Human T-Cell Clones Which Recognize the Surface Protein Antigen of Rickettsia typhi MITCHELl CARL,* SUSMA VAIDYA,1 FU-MEEI

  11. Molecular cloning and nucleotide sequence of cDNA for human liver arginase

    Energy Technology Data Exchange (ETDEWEB)

    Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

    1987-01-01

    Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated lambda hARG6 and lambda hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying lambda hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes.

  12. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence.

    Science.gov (United States)

    D'Aiuto, L; Antonacci, R; Marzella, R; Archidiacono, N; Rocchi, M

    1993-11-01

    We have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed.

  13. Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence

    Energy Technology Data Exchange (ETDEWEB)

    D' Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. (Universita di Bari (Italy)); Antonacci, R. (Instituto Anatomia Umana Normale, Modena (Italy))

    1993-11-01

    The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.

  14. Cloning of non-human primates: the road "less traveled by".

    Science.gov (United States)

    Sparman, Michelle L; Tachibana, Masahito; Mitalipov, Shoukhrat M

    2010-01-01

    Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model.

  15. Cloning of non-human primates: the road “less traveled by”

    Science.gov (United States)

    SPARMAN, MICHELLE L.; TACHIBANA, MASAHITO; MITALIPOV, SHOUKHRAT M.

    2011-01-01

    Early studies on cloning of non-human primates by nuclear transfer utilized embryonic blastomeres from preimplantation embryos which resulted in the reproducible birth of live offspring. Soon after, the focus shifted to employing somatic cells as a source of donor nuclei (somatic cell nuclear transfer, SCNT). However, initial efforts were plagued with inefficient nuclear reprogramming and poor embryonic development when standard SCNT methods were utilized. Implementation of several key SCNT modifications was critical to overcome these problems. In particular, a non-invasive method of visualizing the metaphase chromosomes during enucleation was developed to preserve the reprogramming capacity of monkey oocytes. These modifications dramatically improved the efficiency of SCNT, yielding high blastocyst development in vitro. To date, SCNT has been successfully used to derive pluripotent embryonic stem cells (ESCs) from adult monkey skin fibroblasts. These remarkable advances have the potential for development of human autologous ESCs and cures for many human diseases. Reproductive cloning of nonhuman primates by SCNT has not been achieved yet. We have been able to establish several pregnancies with SCNT embryos which, so far, did not progress to term. In this review, we summarize the approaches, obstacles and accomplishments of SCNT in a non-human primate model. PMID:21404187

  16. Molecular Cloning of TSARG6 Gene Related to Apoptosis in Human Spermatogenic Cells

    Institute of Scientific and Technical Information of China (English)

    Gang LIU; Guang-Xiu LU; Xiao-Wei XING

    2004-01-01

    Beginning from a mouse EST (GenBank accession No. BE644537) which was significantly up-regulated in cryptorchidism and represented a novel gene, we cloned a new gene (GenBank accessionNo. AY138810) which is related to apoptosis in human spermatogenic cells by means of GeneScan programand PCR technology. The gene whose full cDNA length is 1875 bp containing 8 exons and 7 introns islocated in human chromosome lq13.3. Its protein containing 316 amino acid residues is a new member ofHSP40 protein family because the sequence contains the highly conserved J domain which is present in allDna J-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. TSARG6protein displays a 45% identity in a 348-amino acid overlap with DJB5_HUMAN protein. The result ofRT-PCR and Northern blot analysis showed that TSARG6 is specifically expressed in adult testis and thetranscript is 1.8 kb. Based upon all these observations, it is considered that we cloned a new gene whichprobably inhibited human testis spermatogenesis apoptosis.

  17. Sympathy for the Clone: (Post)Human Identities Enhanced by the ‘Evil Science’ Construct and its Commodifying Practices in Contemporary Clone Fiction

    OpenAIRE

    Jimena Escudero Pérez

    2014-01-01

    The manipulation of human DNA in the form of eugenic pursuit, cloning, genetic engineering etc., has become a well-established subject in science fiction for decades now. In our days, this thematic trend is probably the most prolific one when inspiring narratives in popular culture and also constitutes the source of much bioethical debate. A common pattern derived from these practices is that they generate dystopian scenarios where a community is oppressed and abused by scientific means thus ...

  18. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    Energy Technology Data Exchange (ETDEWEB)

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  19. Cloning and adoption: a reply to Levy and Lotz.

    Science.gov (United States)

    Strong, Carson

    2008-02-01

    In previous articles I discussed the ethics of human reproductive cloning, focusing on a possible future scenario in which reproductive cloning can be accomplished without an elevated risk of anomalies to the children who are created. I argued that in such a scenario it would be ethically permissible for infertile couples to use cloning as a way to have genetically related children and that such use should not be prohibited. In 'Reproductive Cloning and a (Kind of) Genetic Fallacy', Neil Levy and Mianna Lotz raise objections to my conclusions. They disagree with the view, for which I argued, that some couples can have defensible reasons for desiring genetically related children. They also offer several new arguments against reproductive cloning, including an argument that it would diminish the number of adoptions, thereby adversely affecting the welfare of children who need to be adopted. In this paper I point out that Levy and Lotz's criticisms misconstrue my arguments and that there are serious problems with their arguments for prohibiting infertile couples from using cloning, including their argument from adoption.

  20. Language and values in the human cloning debate: a web-based survey of scientists and Christian fundamentalist pastors.

    Science.gov (United States)

    Weasel, Lisa H; Jensen, Eric

    2005-04-01

    Over the last seven years, a major debate has arisen over whether human cloning should remain legal in the United States. Given that this may be the 'first real global and simultaneous news story on biotechnology' (Einsiedel et al., 2002, p.313), nations around the world have struggled with the implications of this newly viable scientific technology, which is often also referred to as somatic cell nuclear transfer. Since the successful cloning of Dolly the sheep in 1997, and with increasing media attention paid to the likelihood of a successful human reproductive clone coupled with research suggesting the medical potential of therapeutic cloning in humans, members of the scientific community and Christian fundamentalist leaders have become increasingly vocal in the debate over U.S. policy decisions regarding human cloning (Wilmut, 2000). Yet despite a surfeit of public opinion polls and widespread opining in the news media on the topic of human cloning, there have been no empirical studies comparing the views of scientists and Christian fundamentalists in this debate (see Evans, 2002a for a recent study of opinion polls assessing religion and attitudes toward cloning). In order to further investigate the values that underlie scientists' and Christian fundamentalist leader's understanding of human cloning, as well as their differential use of language in communicating about this issue, we conducted an open-ended, exploratory survey of practicing scientists in the field of molecular biology and Christian fundamentalist pastors. We then analyzed the responses from this survey using qualitative discourse analysis. While this was not necessarily a representative sample (in quantitative terms, see Gaskell & Bauer, 2000) of each of the groups and the response rate was limited, this approach was informative in identifying both commonalities between the two groups, such as a focus on ethical concerns about reproductive cloning and the use of scientific terminology, as well

  1. Limitations on Cloning in Classical Mechanics

    OpenAIRE

    Fenyes, Aaron

    2010-01-01

    In this paper, we show that a result precisely analogous to the traditional quantum no-cloning theorem holds in classical mechanics. This classical no-cloning theorem does not prohibit classical cloning, we argue, because it is based on a too-restrictive definition of cloning. Using a less popular, more inclusive definition of cloning, we give examples of classical cloning processes. We also prove that a cloning machine must be at least as complicated as the object it is supposed to clone.

  2. The Clone Factory

    Science.gov (United States)

    Stoddard, Beryl

    2005-01-01

    Have humans been cloned? Is it possible? Immediate interest is sparked when students are asked these questions. In response to their curiosity, the clone factory activity was developed to help them understand the process of cloning. In this activity, students reenact the cloning process, in a very simplified simulation. After completing the…

  3. Production of Cloned Miniature Pigs Expressing High Levels of Human Apolipoprotein(a in Plasma.

    Directory of Open Access Journals (Sweden)

    Masayuki Ozawa

    Full Text Available High lipoprotein(a [Lp(a] levels are a major risk factor for the development of atherosclerosis. However, because apolipoprotein(a [apo(a], the unique component of Lp(a, is found only in primates and humans, the study of human Lp(a has been hampered due to the lack of appropriate animal models. Using somatic cell nuclear transfer (SCNT techniques, we produced transgenic miniature pigs expressing human apo(a in the plasma. First, we placed the hemagglutinin (HA-tagged cDNA of human apo(a under the control of the β-actin promoter and cytomegalovirus enhancer, and then introduced this construct into kidney epithelial cells. Immunostaining of cells with anti-HA antibody allowed identification of cells stably expressing apo(a; one of the positive clones was used to provide donor cells for SCNT, yielding blastocysts that expressed apo(a. Immunohistochemical analysis of tissue sections and RT-PCR analysis of total RNA from organs of cloned piglet revealed that apo(a is expressed in various tissues/organs including heart, liver, kidney, and intestine. More importantly, a transgenic line exhibited a high level (>400 mg/dL of Lp(a in plasma, and the transgenic apo(a gene was transmitted to the offspring. Thus, we generated a human apo(a-transgenic miniature pig that can be used as a model system to study advanced atherosclerosis related to human disease. The anatomical and physiological similarities between the swine and human cardiovascular systems will make this pig model a valuable source of information on the role of apo(a in the formation of atherosclerosis, as well as the mechanisms underlying vascular health and disease.

  4. Human somatic cell nuclear transfer and reproductive cloning: an Ethics Committee opinion.

    Science.gov (United States)

    2016-04-01

    This document presents arguments that conclude that it is unethical to use somatic cell nuclear transfer (SCNT) for infertility treatment due to concerns about safety; the unknown impact of SCNT on children, families, and society; and the availability of other ethically acceptable means of assisted reproduction. This document replaces the ASRM Ethics Committee report titled, "Human somatic cell nuclear transfer and cloning," last published in Fertil Steril 2012;98:804-7. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  5. Cloning, expression and identification of an isoform of human stromal cell derived factor-1α

    OpenAIRE

    LIANG, YIN-KU; Ping, Wei; BIAN, LIU-JIAO

    2015-01-01

    Human stromal cell derived factor-1α (hSDF-1α), a chemotactic factor of stem cells, regulates inflammation, promotes the mobilization of stem cells and induces angiogenesis following ischemia. Six SDF-1 isoforms, SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1ϕ, which all contain a signal peptide at the N-terminus, have been reported. In the present study a special isoform of hSDF-1α is described that does not contain the N-terminal signal peptide sequence. The hSDF-1α gene was cloned with t...

  6. Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Sakurai, T; Kajitani, M; Kanai, S; Shimazu, T; Shimizu, H; Naruto, M

    1987-07-01

    Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

  7. Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

    Energy Technology Data Exchange (ETDEWEB)

    Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P. (F. Hoffmann-La Roche Ltd., Basel (Switzerland))

    1991-02-15

    A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A){sup +} RNA.

  8. Cloning and Sequence Analysis of Light Variable Region Gene of Anti-human Retinoblastoma Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Zhong; Yongping Li; Shuqi Huang; Bo Ning; Chunyan Zhang; Jianliang Zheng; Guanguang Feng

    2002-01-01

    Purpose: To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.Methods: Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody(McAb)against human retinoblastoma(RB), then transcripted reversely into cDNA with olig-dT primers.The variable region of the light chain (VL) gene fragments was amplified using polymeerase chain reaction(PCR) and further cloned into pGEM(R) -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method.Homologous analysis was done by NCBI BLAST.Results: The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions(FRs)and three complementarity-determining regions (CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.Conclusion: The light chain variable region gene of the McAb against human RB was amplified successfully , which belongs to the Vk9 gene family and utilizes Vk-Jk1 gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.

  9. An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion.

    Science.gov (United States)

    Shutova, Maria V; Surdina, Anastasia V; Ischenko, Dmitry S; Naumov, Vladimir A; Bogomazova, Alexandra N; Vassina, Ekaterina M; Alekseev, Dmitry G; Lagarkova, Maria A; Kiselev, Sergey L

    2016-01-01

    The pluripotency of newly developed human induced pluripotent stem cells (iPSCs) is usually characterized by physiological parameters; i.e., by their ability to maintain the undifferentiated state and to differentiate into derivatives of the 3 germ layers. Nevertheless, a molecular comparison of physiologically normal iPSCs to the "gold standard" of pluripotency, embryonic stem cells (ESCs), often reveals a set of genes with different expression and/or methylation patterns in iPSCs and ESCs. To evaluate the contribution of the reprogramming process, parental cell type, and fortuity in the signature of human iPSCs, we developed a complete isogenic reprogramming system. We performed a genome-wide comparison of the transcriptome and the methylome of human isogenic ESCs, 3 types of ESC-derived somatic cells (fibroblasts, retinal pigment epithelium and neural cells), and 3 pairs of iPSC lines derived from these somatic cells. Our analysis revealed a high input of stochasticity in the iPSC signature that does not retain specific traces of the parental cell type and reprogramming process. We showed that 5 iPSC clones are sufficient to find with 95% confidence at least one iPSC clone indistinguishable from their hypothetical isogenic ESC line. Additionally, on the basis of a small set of genes that are characteristic of all iPSC lines and isogenic ESCs, we formulated an approach of "the best iPSC line" selection and confirmed it on an independent dataset.

  10. Preliminarily functional analysis of a cloned novel human gene ADAM29

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    ADAM is a family of type I integral membrane proteins which are characterized by sharing a disintegrin and metalloprotease domain and involved in many important physiological processes such as fertilization, neurogenesis and inflammatory response. A novel human ADAM gene--ADAM29, which was cloned in our laboratory, is exclusively expressed in human testis and contains a potential fusion domain. A full-length cDNA of ADAM29 was obtained by using multiple-step PCR. Phylogenetic tree of known mammalian ADAMs specifically expressed in testis was reconstructed. Polyclonal antiserum was raised by immunizing the rabbits with sub-peptide of ADAM29 (Leu268-Asp374) as immunogen. The result of immunohistochemical test on human testis showed that ADAM29 is expressed in different stages of spermatogenesis and in interstitial cells. ADAM29 may play a certain role in the signal transduction during the maturation of testis-associated cells.

  11. MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF HUMAN INTERFERON ALPHA2b GENE

    Directory of Open Access Journals (Sweden)

    I. Made Artika

    2013-01-01

    Full Text Available Human alpha Interferons (hIFNα have been shown to have antiviral, antiproliferative and immunomodulatory activities. The human interferon alpha2b (hIFNα2b, is one of the human interferon alpha2 sub variants, naturally synthesized as a polypeptide of 188 amino acid residues, the first 23 residues of which represents a signal peptide. In the present study, the hIFNα2b gene was expressed after being fused with Glutathione S-Transferase (GST gene. The hIFNα2b gene was amplified from human genomic DNA by using a pair of specific primers, cloned into an Escherichia coli expression vector and expressed in E. coli cells under the direction of the tac promoter. The expressed protein was purified using a one-step affinity chromatography column containing immobilized gluthatione-bound resin. The purified protein was shown to react specifically with anti-human-interferon-alpha antibody, confirming that the protein was the human interferon alpha molecule. This strategy has the potential to be used as an alternative mean for production of pure human interferon α proteins for therapeutic purposes and for further studies on their molecular characterization and mechanism of action.

  12. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  13. Actovegin, a non-prohibited drug increases oxidative capacity in human skeletal muscle

    DEFF Research Database (Denmark)

    Søndergård, Stine D; Dela, Flemming; Helge, Jørn W

    2016-01-01

    skeletal muscle. Mitochondrial adaptations like this are also seen after a training program in human subjects. Whether this improvement translates into an ergogenic effect in athletes and thus reiterates the need to include Actovegin on the World Anti-Doping Agency's active list remains to be investigated....

  14. Molecular cloning of a cDNA encoding the human Sm-D autoantigen

    Energy Technology Data Exchange (ETDEWEB)

    Rokeach, L.A.; Haselby, J.A.; Hoch, S.O. (Agouron Institute, La Jolla, CA (USA))

    1988-07-01

    Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. The authors have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg){sub 9} repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen.

  15. [Establishment of stably expressed human RANTES gene in prunella vulgaris cell clone].

    Science.gov (United States)

    Zeng, Qing-Ping; Feng, Li-Ling; Yang, Rui-Yi; Chen, Zhu-Hua

    2003-03-01

    To express interesting human genes in herbal cells for boosting their specific pharmacological activities, RANTES gene cloned from human peripheral blood lymphocyte (PBL) mRNA was introduced into A. tumefaciens strain LBA4404 harboring pAL4404 plasmid via tumor-inducing (Ti) plasmid-derived intermediate expression vector pROKII. In vitro cultured P. vulgaris cells were transformed by leaf-disk cocultivation procedure. Integration of RANTES gene in the genome of transformed cells was confirmed by Southern blotting, and expression of RANTES gene in transformed cells was analyzed by RT-PCR amplification, Western blotting and enzyme-linked immunosorbent assay (ELISA). The peroxidase activity of PBL was utilized as a detection index of cellular chemotropism induction by recombinant RANTES. The results have shown the RANTES gene was integrated in transgenic P. vulgaris cells, and RANTES gene-stably expressed cell clones were available, which could pave the way to obtain transgenic P. vulgaris plants demonstrating specific pharmacological activities.

  16. Physical Characterization of human centromeric regions using transformation-associated recombination cloning technology

    Energy Technology Data Exchange (ETDEWEB)

    Vladimir Larionov, Ph D

    2007-06-05

    A special interest in the organization of human centromeric DNA was stimulated a few years ago when two independent groups succeeded in reconstituting a functional human centromere, using constructs carrying centromere-specific alphoid DNA arrays. This work demonstrated the importance of DNA components in mammalian centromeres and opened a way for studying the structural requirements for de novo kinetochore formation and for construction of human artificial chromosomes (HACs) with therapeutic potential. To elucidate the structural requirements for formation of HACs with a functional kinetochore, we developed a new method for cloning of large DNA fragments for human centromeric regions that can be used as a substrate for HAC formation. This method exploits in vivo recombination in yeast (TAR cloning). In addition, a new strategy for the construction of alphoid DNA arrays was developed in our lab. The strategy involves the construction of uniform or hybrid synthetic alphoid DNA arrays by the RCA-TAR technique. This technique comprises two steps: rolling circle amplification of an alphoid DNA dimer and subsequent assembling of the amplified fragments by in vivo homologous recombination in yeast (Figure 1). Using this system, we constructed a set of different synthetic alphoid DNA arrays with a predetermined sequence varying in size from 30 to 140 kb and demonstrated that some of the arrays are competent in HAC formation. Because any nucleotide can be changed in a dimer before its amplification, this new technique is optimal for identifying the structural requirements for de novo kinetochore formation in HACs. Moreover, the technique makes possible to introduce into alphoid DNA arrays recognition sites for DNA-binding proteins. We have made the following progress on the studying of human centromeric regions using transformation-associated recombination cloning technology: i) minimal size of alphoid DNA array required for de novo kinetochore formation was estimated; ii

  17. Isolation of human minisatellite loci detected by synthetic tandem repeat probes: direct comparison with cloned DNA fingerprinting probes.

    Science.gov (United States)

    Armour, J A; Vergnaud, G; Crosier, M; Jeffreys, A J

    1992-08-01

    As a direct comparison with cloned 'DNA fingerprinting' probes, we present the results of screening an ordered array Charomid library for hypervariable human loci using synthetic tandem repeat (STR) probes. By recording the coordinates of positive hybridization signals, the subset of clones within the library detected by each STR probe can be defined, and directly compared with the set of clones detected by naturally occurring (cloned) DNA fingerprinting probes. The STR probes vary in the efficiency of detection of polymorphic minisatellite loci; among the more efficient probes, there is a strong overlap with the sets of clones detected by the DNA fingerprinting probes. Four new polymorphic loci were detected by one or more of the STR probes but not by any of the naturally occurring repeats. Sequence comparisons with the probe(s) used to detect the locus suggest that a relatively poor match, for example 10 out of 14 bases in a limited region of each repeat, is sufficient for the positive detection of tandem repeats in a clone in this type of library screening by hybridization. These results not only provide a detailed evaluation of the usefulness of STR probes in the isolation of highly variable loci, but also suggest strategies for the use of these multi-locus probes in screening libraries for clones from hypervariable loci.

  18. Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

    Science.gov (United States)

    Baldini, A; Archidiacono, N; Carbone, R; Bolino, A; Shridhar, V; Miller, O J; Miller, D A; Ward, D C; Rocchi, M

    1992-01-01

    We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.

  19. Establishment and identification of fibroblast clones expressing human bone morphogenetic protein 2

    Institute of Scientific and Technical Information of China (English)

    Juan Wang; Weibin Sun; Chun Lu; Guixia Tang

    2005-01-01

    Objective:To establish fibroblasts stably expressing human bone morphogenetic protein 2 (hBMP2). Methods:Eukaryonic expression vector(pcDNA3.1-B2) was transduced into NIH3T3 cells using SofastTM, a new generation cationic polymer gene transfection reagent. The positive cell clones were selected with G418. The stable transfection and expression of BMP2 in the NIH3T3 cells were determined by RT-PCR and immunohistochemical stain. Results: BMP2 mRNA was transcripted and expressed in the transfected NIH3T3 cells. Conclusion: With positive compound transfection, outside human BMP2 gene can be successfully transducted into NIH3T3 cells, which is the key step to induce periodontal cells to osseous phenotypes.

  20. Molecular cloning and chromosomal localization of the ADH7 gene encoding human class IV ({sigma}) ADH

    Energy Technology Data Exchange (ETDEWEB)

    Yokoyama, Hirokazu; Baraona, E.; Lieber, C.S. [Mount Sinai School of Medicine, Bronx, NY (United States)

    1996-01-15

    The ADH7 gene encoding human Class IV ({sigma}) alcohol dehydrogenase (ADH) was cloned from a Caucasian genomic DNA library and characterized. It has nine exons and eight introns that span about 22 kb, and its intron insertion is identical to that of the other ADH genes (ADH1 to ADH5). The nucleotide sequences of the exons encoding 374 amino acids are identical to the previously reported cDNA sequence of {sigma} ADH. Fluorescence in situ hybridization analysis showed that ADH7 is located on human chromosome 4q23-q24, close to the ADH cluster locus (4q21-q25). These data are consistent with the view that Class IV ADH is a member of the ADH family and is phylogenetically close to the other ADHs. 15 refs., 2 figs., 1 tab.

  1. Cloning, functional expression, and characterization of the human prostaglandin E2 receptor EP2 subtype.

    Science.gov (United States)

    Bastien, L; Sawyer, N; Grygorczyk, R; Metters, K M; Adam, M

    1994-04-22

    A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 > iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.

  2. Human phenol sulfotransferase STP2 gene: Molecular cloning, structural characterization, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, C.; Raftogianis, R.; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States)

    1996-05-01

    Sulfonation is an important pathway in the biotransformation of many drugs, xenobiotics, neurotransmitters, and steroid hormones. The thermostable (TS) form of phenol sulfotransferase (PST) preferentially catalyzes the sulfonation of {open_quotes}simple{close_quotes} planar phenols, and levels of activity of TS PST in human tissues are controlled by inheritance. Two different human liver TS PST cDNAs have been cloned that encode proteins with amino acid sequences that are 96% identical. We have determined the structure and chromosomal localization of the gene for one of these two cDNAs, STP2, as a step toward understanding molecular genetic mechanisms involved in the regulation of this enzyme activity in humans. STP2 spans approximately 5.1 kb and contains nine exons that range in length from 74 to 347 bp. The locations of most STP2 exon-intron splice junctions are identical to those of a gene for the thermolabile form of PST in humans, STM; a rat PST gene; a human estrogen ST (EST) gene, STE; and a guinea pig EST gene. The two initial STP2 exons, IA and IB, were identified by performing 5{prime}-rapid amplification of cDNA ends with human liver cDNA as template. Exons IA and IB are noncoding and represent two different human liver TS PST cDNA 5{prime}untranslated region sequences. The two apparent 5{prime}-ons IA and IB, contain no canonical TATA boxes, but do contain CCAAT elements. STP2 was localized to human chromosome 16 by performing the PCR with DNA from NIGMS human/rodent somatic cell hybrids as template. Structural characterization of STP2 will make it possible to begin to study molecular genetic mechanisms involved in the regulation of TS PST activity in human tissues. 63 refs., 7 figs., 1 tab.

  3. [Cloning goat producing human lactoferrin with genetically modified donor cells selected by single or dual markers].

    Science.gov (United States)

    An, Liyou; Yuan, Yuguo; Yu, Baoli; Yang, Tingjia; Cheng, Yong

    2012-12-01

    We compared the efficiency of cloning goat using human lactoferrin (hLF) with genetically modified donor cells marked by single (Neo(r)) or double (Neo(r)/GFP) markers. Single marker expression vector (pBLC14) or dual markers expression vector (pAPLM) was delivered to goat fetal fibroblasts (GFF), and then the transgenic GFF was used as donor cells to produce transgenic goats. Respectively, 58.8% (20/34) and 86.7% (26/30) resistant cell lines confirmed the transgenic integration by PCR. Moreover, pAPLM cells lines were subcultured with several passages, only 20% (6/30) cell lines was observed fluorescence from each cell during the cell passage. Somatic cell nuclear transfer using the donor cells harbouring pBLC14 or pAPLM construct, resulting in a total of 806 reconstructed embryos, a pregnancy rate at 35 d (53.8%, 39.1%) and 60 d (26.9%, 21.7%), and an offspring birth rate (1.9%, 1.4%) with 5 and 7 newborn cloned goats, respectively. Transgene was confirmed by PCR and southern-blot in all cloned offspring. There were no significant differences at the reconstructed embryo fusion rates, pregnancy rates and the birth rate (P > 0.05) between single and double markers groups. The Neo(r)/GFP double markers could improve the reliability for accurately and efficiently selecting the genetically modified donor cells. No adverse effect was observed on the efficiency of transgenic goat production by SCNT using somatic cells transfected with double (Neo(r)/GFP) markers vector.

  4. cDNA cloning of human myeloperoxidase: decrease in myeloperoxidase mRNA upon induction of HL-60 cells

    Energy Technology Data Exchange (ETDEWEB)

    Weil, S.C.; Rosner, G.L.; Reid, M.S.; Chisholm, R.L.; Farber, N.M.; Spitznagel, J.K.; Swanson, M.S.

    1987-04-01

    Myeloperoxidase (MPO), the most abundant neutrophil protein, is a bacteriocidal component of the primary granules and a critical marker in distinguishing acute myelogenous leukemia from acute lymphoid leukemia. A cDNA clone for human MPO was isolated by immunologic screening of human hematopoietic lambdagt11 expression vector libraries with specific anti-MPO antibody. The identity of the cDNA clone was confirmed by finding that (i) epitope-selected antibody against this clone recognizes purified MPO and MPO in human promyelocytic (HL-60) cell lysates by immunoblot analysis, and that (ii) hybrid section of HL-60 mRNA with this cDNA clone and translation in vitro results in the synthesis of an 80-kDa protein recognized by the anti-MPO antiserum. RNA blot analysis with this MPO cDNA clone detects hybridization to two polyadenylylated transcripts of approx. = 3.6 and approx. = 2.9 kilobases in HL-60 cells. No hybridization is detected to human placenta mRNA. Upon induction of HL-60 cells to differentiate by incubation for 4 days with dimethyl sulfoxide, a drastic decrease in the hybridization intensity of these two bands is seen. This is consistent with previous data suggesting a decrease in MPO synthesis upon such induction of these cells. The MPO cDNA should be useful for further molecular and genetic characterization of MPO and its expression and biosynthesis in normal and leukemic granulocytic differentiation.

  5. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A L; Cubellis, M V; Masucci, M T

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis...

  6. Can hospitals prohibit euthanasia? An analysis from a European human rights perspective.

    Science.gov (United States)

    Tack, Sylvie

    2011-06-01

    At present, in four European countries euthanasia and/ or physician assisted suicide (PAS) are tolerated under strict legal conditions. However, in practice these patient groups are often deprived of the possibility to undergo such decisions. Particularly Catholic health care institutions have developed policies which restrict the internal application of the law. Yet, the legitimacy of such policies is questionable. From a European human rights perspective it can be defended that the freedom of association allows hospitals to develop policies elaborating their ethical stances on euthanasia and PAS. However, to respect the patient's right to self-determination the concerned hospitals should at least inform current and future patients about the restrictive policy and deal carefully with euthanasia and PAS requests. If a patient's wish remains seriously incompatible with the ethical stances of the hospital, at least reasonable and attainable alternatives (such as a referral to a tolerant regional hospital) should be offered.

  7. Ethical issues in cloning.

    Science.gov (United States)

    Satris, S

    2000-01-01

    There is great public concern with the ethics of human cloning. This paper briefly examines some of what I identify as pseudo-problems or myths associated with cloning, and some of the more substantial ethical concerns.

  8. Just another reproductive technology? The ethics of human reproductive cloning as an experimental medical procedure.

    Science.gov (United States)

    Elsner, D

    2006-10-01

    Human reproductive cloning (HRC) has not yet resulted in any live births. There has been widespread condemnation of the practice in both the scientific world and the public sphere, and many countries explicitly outlaw the practice. Concerns about the procedure range from uncertainties about its physical safety to questions about the psychological well-being of clones. Yet, key aspects such as the philosophical implications of harm to future entities and a comparison with established reproductive technologies such as in vitro fertilisation (IVF) are often overlooked in discussions about HRC. Furthermore, there are people who are willing to use the technology. Several scientists have been outspoken in their intent to pursue HRC. The importance of concerns about the physical safety of children created by HRC and comparisons with concerns about the safety of IVF are discussed. A model to be used to determine when it is acceptable to use HRC and other new assisted reproductive technologies, balancing reproductive freedom and safety concerns, is proposed. Justifications underpinning potential applications of HRC are discussed, and it is determined that these are highly analogous to rationalisations used to justify IVF treatment. It is concluded that people wishing to conceive using HRC should have a prima facie negative right to do so.

  9. Cloning, structural organization, and chromosomal mapping of the human phenol sulfotransferase STP2 gene

    Energy Technology Data Exchange (ETDEWEB)

    Gaedigk, A.; Beatty, B.G.; Grant, D.M. [Hospital for Sick Children, Toronto, Ontario (Canada)

    1997-03-01

    Phenol- and monoamine-metabolizing sulfotransferases (STP and STM, respectively) are members of a superfamily of enzymes that add sulfate to a variety of xenobiotics and endobiotics containing hydroxyl or amino functional groups. To characterize related sulfotransferase genes further, we used extra-long PCR (XL-PCR) to generate three distinct sizes of amplification products from human genomic DNA or from genomic phage library clones, each of which contained sulfotransferase gene sequences. One of the PCR fragments contained a new sulfotransferase gene, STP2, corresponding to a recently published cDNA clone that encodes a sulfotransferase with catalytic specificity distinct from that of the previously described STP1 and STM. Additional upstream sequence information was obtained using a second STP2-specific XL-PCR-based approach. The STP2 gene is composed of eight exons and seven introns, with exon sizes ranging from 95 to 181 bp. Protein-coding exon lengths and locations of the splice junctions were identical to those in both the STM gene and an STP2 gene published independently by another group recently. The STP2 gene maps to a chromosomal location (16p11.2-p1.2) that is the same as that previously determined for both STP1 and STM. The characterization of the STP2 gene provides further insight into the organization, regulation, and multiplicity of the sulfotransferase supergene family. 27 refs., 3 figs.

  10. Identification of novel human WW domain-containing proteins by cloning of ligand targets.

    Science.gov (United States)

    Pirozzi, G; McConnell, S J; Uveges, A J; Carter, J M; Sparks, A B; Kay, B K; Fowlkes, D M

    1997-06-06

    A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-alpha, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.

  11. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells.

    OpenAIRE

    1991-01-01

    The human embryonal carcinoma cell line NEC14 can be induced to differentiate by the addition of 10(-2)M N,N'-hexamethylene-bis-acetamide (HMBA). A subtractive cDNA library specific to undifferentiated NEC14 cells was constructed using oligo(dT)30-Latex and polymerase chain reaction (PCR). The method was designed to improve the efficiency of subtraction and the enrichment of cDNA clones corresponding to low abundance mRNAs. The single strand of cDNA was made from mRNA prepared from the HMBA-t...

  12. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand.

    Science.gov (United States)

    Thaipadungpanit, Janjira; Wuthiekanun, Vanaporn; Chierakul, Wirongrong; Smythe, Lee D; Petkanchanapong, Wimol; Limpaiboon, Roongrueng; Apiwatanaporn, Apichat; Slack, Andrew T; Suputtamongkol, Yupin; White, Nicholas J; Feil, Edward J; Day, Nicholas P J; Peacock, Sharon J

    2007-10-31

    A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection. Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.

  13. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand.

    Directory of Open Access Journals (Sweden)

    Janjira Thaipadungpanit

    2007-10-01

    Full Text Available A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown.A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34 accounted for 77 (76% of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01. ST34 represented 17/24 (71% of human isolates from other Thai provinces, and 7/8 (88% rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection.Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.

  14. Cloning and Expression of Recombinant Human Thymosin in Yeast Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Cao Junxia(曹俊霞); Jin Liji; Duan Yanlong; An Lijia

    2003-01-01

    The gene of human thymosin alpha 1(hT(1)was synthesised according to favorite codons of Pichia pastoris by PCR. N-terminal 28 amino acid residues of 40S ribosomal protein (RP), S24E that is N-acetylserine were replaced by hT(1 for the constitution of hT(1-RP fusion gene in order to express acetyllated thymosin α1. And also,the Asn-Gly bond was designed to faciliate isolation of the target protein.The fusion gene was cloned into the expression vector, pPIC/9K. The constructs were transformed into HIS4 mutant strain GS115 by electroporation. Both SDS-PAGE analysis and Western blot analysis indicated that the fusion protein was expressed successfully.

  15. Isolation and characterization of cloned cDNAs as encoding human liver chlordecone reductase

    Energy Technology Data Exchange (ETDEWEB)

    Winters, C.J.; Molowa, D.T.; Guzelian, P.S. (Medical College of Virginia, Richmond (USA))

    1990-01-30

    Chlordecone (Kepone), a toxic organochlorine pesticide, undergoes bioreduction to chlorodecone alcohol in human liver. This reaction is controlled by a cytosolic enzyme, chlordecone reductase (CDR), which may be of the aldo-keto reductase family of xenobiotic metabolizing enzymes. To further investigate the primary structure and expression of CDR, the authors screened a library of human liver cDNAs cloned in the expression vector {lambda}gt11 and isolated an 800 bp cDNA that directed synthesis of a fusion protein recognized by polyclonal anti-CDR antibodies. Using this cDNA as a probe, they screened two human liver cDNA libraries and found several 1.2-kb cDNAs which would code for polypeptide with 308 residues (35.8 kDa). However, a similar full-length cDNA, possibly the transcript of a pseudogene, contained an in-frame nonsense codon. The deduced protein sequence of CDR showed 65% similarity to the primary structure of human liver aldehyde reductase and 66% similarity to the inferred protein sequence of rat lens aldose reductase. A search of GenBank revealed significant nucleotide similarity to a cDNA coding for bovine lung prostaglandin f synthase and to a partial cDNA coding for frog lens {rho}-crystallin. RNA from adult but not fetal human liver, and from the human hepatoma cell-line Hep G2, contained major (1.6 kb) and minor (2.8 kb) species hybridizable to a CDR cDNA. The relative amounts of these RNAs varied markedly among nine subjects. From this initial description of the nucleotide sequence for a human carbonyl reductase, they conclude that CDR and several related enzymes are part of a novel multigene family involved in the metabolism of such xenobiotics as chlordecone and possibly endogenous substrates.

  16. Ultra high performance liquid chromatography tandem mass spectrometry determination and profiling of prohibited steroids in human biological matrices. A review.

    Science.gov (United States)

    Gosetti, Fabio; Mazzucco, Eleonora; Gennaro, Maria Carla; Marengo, Emilio

    2013-05-15

    The use of doping agents, once restricted to professional athletes, has nowadays become a problem of public health, since it also concerns young people and non-competing amateurs in different sports. The use is also diffused in social life for improving physical appearance and enhancing performance and even dietary supplements assumed to improve performance often contain anabolic steroids. While decades ago the so-called "classical doping agents" (like stimulants and narcotics) were used, to-day anabolic steroids are more widely diffused. Anabolic steroids are synthetic substances prepared by introducing modifications in the molecular structure of testosterone, the main natural androgenic anabolic steroid that forms in testes interstitial cells. The first report concerning the use of anabolic steroids by an athlete who searched for increased weight and power dates 1954. In 1974 the misuse of anabolic steroids in sports was banned by the International Olympic Committee and control tests were implemented in 1976 Montreal Olympic Games through radioimmunoassay analysis: the technique, however, only allows for unspecific detection of a limited number of exogenous steroids. Over the years, always new doping substances are synthesized and, as a consequence, the list of prohibited compounds is continuously updated and new suitable analytical methods for their detection and determination in biological matrices are continuously required. In doping control analysis the knowledge of steroid metabolism pathway in human body is of primary importance and the analytical methods must permit the simultaneous detection and determination not only of the forbidden precursor agents but also of their metabolites. In addition, the potential presence and amount in the biological samples of species that can interfere in the analysis should be evaluated. Also the several anabolic steroids, specifically designed to circumvent doping control, put on the market have been incorporated in the

  17. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  18. Molecular cloning and characterization of the human beta-like globin gene cluster.

    Science.gov (United States)

    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  19. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, Morten Hanefeld; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  20. High efficiency ex vivo cloning of antigen-specific human effector T cells.

    Directory of Open Access Journals (Sweden)

    Michelle A Neller

    Full Text Available While cloned T cells are valuable tools for the exploration of immune responses against viruses and tumours, current cloning methods do not allow inferences to be made about the function and phenotype of a clone's in vivo precursor, nor can precise cloning efficiencies be calculated. Additionally, there is currently no general method for cloning antigen-specific effector T cells directly from peripheral blood mononuclear cells, without the need for prior expansion in vitro. Here we describe an efficient method for cloning effector T cells ex vivo. Functional T cells are detected using optimised interferon gamma capture following stimulation with viral or tumour cell-derived antigen. In combination with multiple phenotypic markers, single effector T cells are sorted using a flow cytometer directly into multi-well plates, and cloned using standard, non antigen-specific expansion methods. We provide examples of this novel technology to generate antigen-reactive clones from healthy donors using Epstein-Barr virus and cytomegalovirus as representative viral antigen sources, and from two melanoma patients using autologous melanoma cells. Cloning efficiency, clonality, and retention/loss of function are described. Ex vivo effector cell cloning provides a rapid and effective method of deriving antigen-specific T cells clones with traceable in vivo precursor function and phenotype.

  1. Cloning of NHE-1 gene fragment from human lung cancer cells and construction of its antisense expression vector

    Institute of Scientific and Technical Information of China (English)

    WU Guo-ming; HUANG Gui-jun; QIAN Gui-sheng

    2001-01-01

    To clone the partial sequence of Na+/H+ exchanger-1 (NHE-1) gene of human lung cancer cells and insert it reversely into the multiclone site of pLXSN in order to construct an antisense expression vector for tumor gene therapy in vivo. Methods: With use of the upstream and downstream primers containing Bam H I and EcoR I in their 5' ends respectively, a partial sequence of the first exon of NHE-1 gene was cloned in a length of 454 bp from genomic DNA of human lung cancer cell A549 with PCR method. The product was then directionally and reversely insert into the multiclone site of pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophoresis and DNA sequencing. Results: The cloned fragment was 461 bp in length and successfully ligated to pLXSN with the identification by agarose gel electrophoresis. DNA sequencing confirmed that the fragment cloned and inserted into the vector was identical with the targeted one. Conclusion: The targeted fragment is successfully cloned and reversely inserted into pLXSN in our experiment. The antisense expression vector ofNHE-1, pNHE- 1, was constructed successfully.

  2. T-cell libraries allow simple parallel generation of multiple peptide-specific human T-cell clones

    Science.gov (United States)

    Theaker, Sarah M.; Rius, Cristina; Greenshields-Watson, Alexander; Lloyd, Angharad; Trimby, Andrew; Fuller, Anna; Miles, John J.; Cole, David K.; Peakman, Mark; Sewell, Andrew K.; Dolton, Garry

    2016-01-01

    Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6 weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer. PMID:26826277

  3. Cloning, expression, and functional analysis of human dopamine D1 receptors

    Institute of Scientific and Technical Information of China (English)

    Wan-chun SUN; Lei JIN; Yan CAO; Li-zhen WANG; Fan MENG; Xing-zu ZHU

    2005-01-01

    Aim: To construct an HEK293 cell line stably expressing human dopamine D1 receptor (D1R). Methods: cDNA was amplified by RT-PCR using total RNA from human embryo brain tissue as the template. The PCR products were subcloned into the plasmid pcDNA3 and cloned into the plasmid pcDNA3.1. The cloned D1R cDNA was sequenced and stably expressed in HEK293 cells. Expression of D1R in HEK293 cells was monitored by the [3H]SCH23390 binding assay. The function of D1R was studied by the cAMP accumulation assay, CRE-SEAP reporter gene activity assay, and intracellular calcium assay. Results: An HEK293 cell line stably expressing human D1R was obtained. A saturation radioligand binding experiment with [3H]SCH23390 demonstrated that the Kd and Bmax values were 1.5±0.2 nmol/L and 2.94±0.15 nmol/g of protein, respectively. In the[3H]SCH23390 competition assay, D1R agonist SKF38393 displaced[3H]SCH23390 with an IC50 value of 2.0 (1.5-2.8) μmol/L. SKF38393 increased the intracellular cAMP level and CRE-SEAP activity through D1R expressed in HEK293 cells in a concentration-dependent manner with an EC50 value of 0.25(0.12-0.53) μmol/L and 0.39 (0.27-0.57) μmol/L at 6 h/0.59 (0.22-1.58) μmol/L at 12 h, respectively. SKF38393 also increased the intracellular calcium level in a concentration-dependent manner with EC50 value of 27 (8.6-70) nmol/L.Conclusion: An HEK293 cell line stably expressing human D1R was obtained successfuly. The study also demonstrated that the CRE-SEAP activity assay could be substituted for the cAMP accumulation assay for measuring increase in cAMP levels. Thus, both intracellular calcium measurements and the CRE-SEAP activity assay are suitable for high-throughput screening in drug research.

  4. Popular Theatre for Science Engagement: Audience Engagement with Human Cloning Following a Production of Caryl Churchill's "A Number"

    Science.gov (United States)

    Donkers, Martina; Orthia, Lindy A.

    2016-01-01

    Research into the role of fiction in engaging people with science is a growing area, but a little studied medium in this respect is "popular theatre," or non-pedagogic theatre that exists primarily as a work of art. This study investigated audience engagement with human cloning issues after seeing a performance of Caryl Churchill's 2002…

  5. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, A A; Sheppard, P O

    2001-01-01

    predicted to encode an additional subtype. The full length coding regions of mouse mGprc5d and human GPRC5D were cloned and shown to contain predicted open reading frames of 300 and 345 amino acids, respectively. GPRC5D has seven putative transmembrane segments and is expressed in the cell membrane...

  6. A human CD5+ B cell clone that secretes an idiotype-specific high affinity IgM monoclonal antibody.

    NARCIS (Netherlands)

    R.W.J. van der Heijden (Roger); H. Bunschoten; A.C. Hoek (Aad); J. van Es (Johan); M. Punter; A.D.M.E. Osterhaus (Albert); F.G.C.M. Uytdehaag (Fons)

    1991-01-01

    textabstractWe previously demonstrated the occurrence of a naturally arisen human anti-idiotypic B cell clone, that we transformed with EBV (EBV383). We show evidence that EBV383 not only expresses the CD5 surface Ag, but also contains the 2.7-kb mRNA transcript encoding this protein. In addition, w

  7. Establishment and Analysis of Cancer Stem-Like and Non-Cancer Stem-Like Clone Cells from the Human Colon Cancer Cell Line SW480.

    Science.gov (United States)

    Takaya, Akari; Hirohashi, Yoshihiko; Murai, Aiko; Morita, Rena; Saijo, Hiroshi; Yamamoto, Eri; Kubo, Terufumi; Nakatsugawa, Munehide; Kanaseki, Takayuki; Tsukahara, Tomohide; Tamura, Yasuaki; Takemasa, Ichiro; Kondo, Toru; Sato, Noriyuki; Torigoe, Toshihiko

    2016-01-01

    Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

  8. Cloning, tissue expression pattern, and chromosome localization of human protein kinase Bγ gene

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows that PKB undergoes activation in some human tumors and is involved in Ras pathway, which implies that PKB can trigger a pathway to induce oncogenic transformation. A nucleotide sequence of mouse Pkb? was used as a probe to screen homolog in a human liver cDNA library. A fragment of 1998 bp containing a 1440 bp ORF encoding 479 amino acid residues was obtained. Then the 3'-terminal of this fragment was extended to 2788 bp by 'electronic walking' screening, and the extended fragment was confirmed by PCR amplification. The protein deduced by the gene had a high identity of 83% and 78% to the human PKBγ and γ, respectively, and was designated as human PKB?. Northern hybridization detected two equally expressed transcripts of 8.5 and 6.5 kb in length in all 16 human tissues tested, with the highest expression level in brain, and lower levels with variation in the other tissues. By RH mapping, the PKBγ was placed on chromosome 1q43, between markers D1S304 and D1S2693. It is a valuable clue for cloning the candidate genes related to prostate cancer; Arrhythmogenic Right Ventricular Dysplasia (ARVD); Chediak-Higashi, NK cell Deficiency (CHS); and Hypoparathyrodism with Short Stature, Mental Retardation and Seizures which have already been mapped in this chromosomal region.

  9. 45 CFR 1110.3 - Discrimination prohibited.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Discrimination prohibited. 1110.3 Section 1110.3... HUMANITIES GENERAL NONDISCRIMINATION IN FEDERALLY ASSISTED PROGRAMS § 1110.3 Discrimination prohibited. (a... from participation in, be denied the benefits of, or be otherwise subjected, to discrimination under...

  10. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  11. Molecular cloning and expression of a novel human cDNA containing CAG repeats.

    Science.gov (United States)

    Takeuchi, T; Chen, B K; Qiu, Y; Sonobe, H; Ohtsuki, Y

    1997-12-19

    A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.

  12. Cloning and characterization of the human integrin β6 gene promoter.

    Directory of Open Access Journals (Sweden)

    Mingyan Xu

    Full Text Available The integrin β6 (ITGB6 gene, which encodes the limiting subunit of the integrin αvβ6 heterodimer, plays an important role in wound healing and carcinogenesis. The mechanism underlying ITGB6 regulation, including the identification of DNA elements and cognate transcription factors responsible for basic transcription of human ITGB6 gene, remains unknown. This report describes the cloning and characterization of the human ITGB6 promoter. Using 5'-RACE (rapid amplification of cDNA ends analysis, the transcriptional initiation site was identified. Promoter deletion analysis identified and functionally validated a TATA box located in the region -24 to -18 base pairs upstream of the ITGB6 promoter. The regulatory elements for transcription of the ITGB6 gene were predominantly located -289 to -150 from the ITGB6 promoter and contained putative binding sites for transcription factors such as STAT3 and C/EBPα. Using chromatin immunoprecipitation assays, this study has demonstrated, for the first time, that transcription factors STAT3 and C/EBPα are involved in the positive regulation of ITGB6 transcription in oral squamous cell carcinoma cells. These findings have important implications for unraveling the mechanism of abnormal ITGB6 activation in tissue remodeling and tumorigenesis.

  13. Molecular basis of essential fructosuria: molecular cloning and mutational analysis of human ketohexokinase (fructokinase).

    Science.gov (United States)

    Bonthron, D T; Brady, N; Donaldson, I A; Steinmann, B

    1994-09-01

    Essential fructosuria is one of the oldest known inborn errors of metabolism. It is a benign condition which is believed to result from deficiency of hepatic fructokinase (ketohexokinase, KHK, E.C.2.7.1.3). This enzyme catalyses the first step of metabolism of dietary fructose, conversion of fructose to fructose-1-phosphate. Despite the early recognition of this disorder, the primary structure of human KHK and the molecular basis of essential fructosuria have not been previously defined. In this report, the isolation and sequencing of full-length cDNA clones encoding human ketohexokinase are described. Alternative mRNA species and alternative KHK isozymes are produced by alternative polyadenylation and splicing of the KHK gene. The KHK proteins show a high level of sequence conservation relative to rat KHK. Direct evidence that mutation of the KHK structural gene is the cause of essential fructosuria was also obtained. In a well-characterized family, in which three of eight siblings have fructosuria, all affected individuals are compound heterozygotes for two mutations Gly40Arg and Ala43Thr. Both mutations result from G-->A transitions, and each alters the same conserved region of the KHK protein. Neither mutation was seen in a sample of 52 unrelated control individuals. An additional conservative amino acid change (Val49IIe) was present on the KHK allele bearing Ala43Thr.

  14. The cloning and preliminarily functional analysis of the human neurotrimin gene

    Institute of Scientific and Technical Information of China (English)

    LIU; Jin; LI; Guangtao; PENG; Xiaozhong; LIU; Bingyan; YIN

    2004-01-01

    Proteins of the immunoglobulin superfamily (IgSF) are involved in a variety of specific cell-cell interactions in the developing nervous system. To identify and characterize new members of this protein family in human nervous system, we screen the human fetal brain cDNA library and isolate a full-length cDNA clone which contains a 1032 bp open reading frame encoding a protein of 344 amino acids. Sequence analysis reveals that it is a glycoprotein comprised of three C2-like immunoglobulin domains and is anchored to the plasma membrane via a post-translationally attached glycosyl-phosphatidylinositol (GPI) moiety. The protein shows high sequence similarity to the rat Ntm (97%), so we term it human neurotrimin (NTM). Northern blot analysis reveals that (HUMAN)NTM has three different transcripts with the length of 3.2 kb, 4.0 kb and 9.0 kb respectively. It has a wider expression pattern than that of (RAT) Ntm. Notably, the expression of NTM in fetal brain is higher than that in mature brain and is stronger in nervous tumors than that in normal brain tissues. We insert an HA epitope tag between the third Ig-like domain of NTM and the site of GPI attachment, then construct it into the eukaryotic expression vector pcDNA3.1+/Zeocin. The pcDNA3.1-HA-NTM is transfected into the Chinese Hamster Ovary (CHO) cells. The results demonstrate that HA-NTM is expressed on the surface of CHO cells and could strengthen the aggregation of CHO-NTM cells.

  15. Cloning and expression of a cDNA encoding human sterol carrier protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia (United States)); Billheimer, J.T. (E.I. DuPont de Nemours, Inc., Wilmington, DE (United States))

    1991-01-15

    The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP{sub 2}). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP{sub 2} amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP{sub 2}. The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A){sup +} RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP{sub 2} gene in the human genome or that the SCP{sub 2} gene is very large. Coexpression of the SCP{sub 2} cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP{sub 2} plays a role in regulating steroidogenesis, among other possible functions.

  16. Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs.

    Science.gov (United States)

    Chumakov, I M; Zabarovsky, E R; Prassolov, V S; Mett, V L; Kisselev, L L

    1982-01-01

    A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage lambda Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.

  17. Why Clone?

    Science.gov (United States)

    ... How might cloning be used in medicine? Cloning animal models of disease Much of what researchers learn ... issue of the genetic reshuffling that happensduring sexual reproduction and simply clone our drug-producing cow. Cloning ...

  18. Cloning and expression analysis of human reticulon 4c cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    RTNs (reticulons) is a gene family related to the growth and differentiation of neuroendocrine cell. This family is composed of several members such as RTN1, RTN2 and RTN3. RTN1 and RTN2 have been proved to have 3 transcripts with different length. Because the RTN1c cDNA was involved in the sologenesis of small cell lung carcinoma (SCLC), it was selected as a bioinformatic probe to clone novel members of RTN family with the electric hybridization assistant new-gene cloning method (EHAC). A 1677-bp cDNA was identified from human brain cDNA library. The cDNA contains an intact open reading frame (ORF) which encodes a protein of 199 amino acids. This deduced protein is highly homologous to RTN1c, RTN2c and RTN3 with identities of 64.4%, 45.8% and 50.0% respectively. This new gene was named RTN4c (GenBank accession number: AF087901). Northern hybridization showed that the full length of RTN4c transcript is about 1.8 kb. It is hardly expressed in heart, placenta, lung, spleen, thymus, testis, ovary, small intestine and peripheral blood white cells; but it is highly expressed in the tissues of skeletal muscle, brain, liver and kidney, and less expressed in the pancreas, prostate and colon. Furthermore, Northern results also showed that there is a 2.3 kb transcript expressed in 14 tissues except liver and skeletal muscle; while another 5.0 kb transcript in brain, skeletal muscle and testis. By the electric hybridization walking, we obtained two full-length contigs with a length of 4632 and 2235 bp respectively. The former encodes a protein with 1192 amino acids and was defined as RTN4a; the latter encodes another protein with 373 amino acids, and was named RTN4b. The RTN4 gene was mapped to human chromosome 2p14-p13 region by the radiation hybridization (RH).

  19. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire].

    Science.gov (United States)

    Guillot-Chene, P; Lebecque, S; Rigal, D

    2009-05-01

    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

  20. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    GU Fu; LI YuYang; L(U) Hong; YOU Chun; LIU JianPing; CHEN Ao; YU Yao; WANG Xiang; WAN DaFang; GU JianRen; YUAN HanYing

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recombination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepatocellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepatocarcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E.coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chromatography in an FPLC system. The analysis using isotope α-32p-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  1. Cloning, expression and characterization of human tissue-specific DNA polymerase λ2

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    DNA polymerase (POL) λ plays an important role during DNA repair and DNA nonhomologous recom-bination processes. A novel POL λ variant was cloned from a human liver cDNA library and named POL λ2 (GenBank Accession No. AY302442). POL λ2 has 2206 base pairs in length with an open reading frame of 1452 base pairs encoding a 482-amino-acids protein. Bioinformatics analysis reveals that POL λ2 spans 7.9 kb on human chromosome 10q24 and is composed of 8 exons and 7 introns. It has the specific domain of DNA polymerase X family-POL Xc at the C-terminus and BRCT domain at the N-terminus. POL λ2 was localized predominantly in nucleus in transfected L0-2 cells. It was expressed abundantly in liver and testis, weakly in ovary, and undetectably in other tested human tissues. In comparison with the expression ratio between POL λ and POL λ2 in normal liver tissues and hepato-cellular carcinoma (HCC) adjacent tissues, the ratio was aberrant in 80% of those 15 HCC specimens examined due to the up-regulated expression of POL λ. This abnormality might be involved in hepato-carcinogenesis. The recombinant POL λ2 with His-tag was expressed as a soluble active protein in E. coli BL21 (DE3)CONDON Plus and purified by Ni-NTA resin and then desalted by Superdex-75 chro-matography in an FPLC system. The analysis using isotope α-32P-dCTP incorporation in vitro showed that the purified recombinant POL λ2 exhibited DNA polymerase activity.

  2. Cloning, Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells.

    Science.gov (United States)

    Shrestha, Pravesh; Yun, Ji-Hye; Kim, Woo Taek; Kim, Tae-Yoon; Lee, Weontae

    2016-03-01

    A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1-240) and truncated (residues 19-240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.

  3. Cloning,sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

    Institute of Scientific and Technical Information of China (English)

    ZHANGJINSONG; MINGYEH

    1994-01-01

    The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.

  4. The cloning, genomic organization and tissue expression profile of the human DLG5 gene

    Directory of Open Access Journals (Sweden)

    Gibbs Richard A

    2002-02-01

    Full Text Available Abstract Background Familial atrial fibrillation, an autosomal dominant disease, was previously mapped to chromosome 10q22. One of the genes mapped to the 10q22 region is DLG5, a member of the MAGUKs (Membrane Associated Gyanylate Kinase family which mediates intracellular signaling. Only a partial cDNA was available for DLG5. To exclude potential disease inducing mutations, it was necessary to obtain a complete cDNA and genomic sequence of the gene. Methods The Northern Blot analysis performed using 3' UTR of this gene indicated the transcript size to be about 7.2 KB. Using race technique and library screening the entire cDNA was cloned. This gene was evaluated by sequencing the coding region and splice functions in normal and affected family members with familial atrial fibrillation. Furthermore, haploid cell lines from affected patients were generated and analyzed for deletions that may have been missed by PCR. Results We identified two distinct alternately spliced transcripts of this gene. The genomic sequence of the DLG5 gene spanned 79 KB with 32 exons and was shown to have ubiquitous human tissue expression including placenta, heart, skeletal muscle, liver and pancreas. Conclusions The entire cDNA of DLG5 was identified, sequenced and its genomic organization determined.

  5. Cloning and expression of MXR7 gene in human HCC tissue

    Institute of Scientific and Technical Information of China (English)

    Xue Ping Zhou; Hong Yang Wang; Guang Shun Yang; Zheng Jun Chen; Bao An Li; Meng Chao Wu

    2000-01-01

    AIM To clone and identify the whole cDNA of MXR7 gene and to find out its expression in human HCC, and normal tissues.METHODS The DNA primers were designed and synthesized according to the whole cDNA sequence of MXR7 gene. The cDNA of human HCC was taken as the template while the cDNA of MXR7 gene was synthesized by polymerase chain reaction (PCR). Recombinant DNA conforming to reading frame was constructed by connecting purified PCR product of the cDNA of MXR7 gene with expression vector pGEX-5X-1 of fusion protein. The plasmid MXR7/pGEX-5X-1 was identified by sequencing. Using 32P labeled MXR7 cDNA as probe, MXR7 mRNA expression was detected by Northern blot analysis in 12 different human normal tissues, 7 preoperatively untreated non-liver tumor tissues, 30 preoperatively untreated HCC, the paracancerous liver tissues and 12 normal liver tissues samples.RESULTS Restriction enzyme and sequence analysis confirmed that the insertion sequence in vector pGEX-5X-1 was the same as the cDNA sequence of MXR7 gene. Northern blot analysis showed no expression of MXR7 mRNA in 12 kinds of normal human tissues including liver, 7 tumor tissues in other sites and 12 normal liver tissues, the frequencies of MXR7 mRNA expression in HCC and paracancerous liver tissues were 76.6% and 13.3%, respectively.The frequency of MXR7 mRNA expression in HCC without elevation of serum AFP and in HCC <5cm was 90% (9/10) and 83.3% (5/6),respectively.CONCLUSION MXR7 mRNA is highly expressed in human HCC, which is specific and occurs at an early stage of HCC, suggesting MXR7 mRNA can be a tumor biomarker for HCC. The detection of MXR7 mRNA expression in the biopsied liver tissue is helpful in discovering early subclinical liver cancer in those with negative serum AFP.

  6. Detection of cancer clones in human gastric adenoma by increased DNA-instability and other biomarkers

    Directory of Open Access Journals (Sweden)

    A Sun

    2009-06-01

    Full Text Available An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30°C for 20 min (DNA-instability test has been used as a marker of malignancy. The test was applied to bioptic tissues of human gastric polyp assessed histopathologically as foveolar hyperplastic polyp (13 cases, mild (58 cases, moderate (86 cases, and severe (20 cases dysplasia, and adenocarcinomas (14 cases. The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor (DFF45, and basic fibroblast growth factor (bFGF. The DNA-instability test was positive in 14 (100% adenocarinoma cases, 20 (100% severe dysplasia cases, 52 (60.5% moderate dysplasia cases, and 12 (20.7% mild dysplasia cases, indicating malignancy. All foveolar hyperplastic polyps were negative to the DNA-instability testing. Furthermore, the percentage of glands positive in the DNA-instability test steadily increased in going from mild (10%, to moderate (40%, to severe (100% dysplasia, and adenocarcinoma (100%. All other biological markers tested in the present study showed significantly higher values in the adenoma glands, being positive to DNA-instability testing, irrespective of the dysplasia grade, as compared to those in the adenoma glands that were negative to DNA-instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. These results indicate that cancer cell clones are already present at the adenoma stages showing a positive DNA-instability test, enhanced proliferative activity, p53 mutation, induction of DFF45 and bFGF. These factors allow cancer cell proliferation, producing heterogeneous subclones due to DNA-instability, enhancing their survival by escaping apoptosis, and providing abundant nutrients during the early-stage progression of gastric cancer. Based on these findings, we herein propose the

  7. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    DEFF Research Database (Denmark)

    Roldan, A.L.; Cubellis, M.V.; Masucci, M.T.

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis...... fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells...

  8. Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1

    NARCIS (Netherlands)

    Nielsen, Peter A; Beahm, Derek L; Giepmans, Ben N G; Baruch, Amos; Hall, James E; Kumar, Nalin M

    2002-01-01

    A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.

  9. Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L-cells.

    NARCIS (Netherlands)

    M.J. Brown (Morris); A.W.R. Tyrrell; C.G. Chapman; J.E. Carey (Janet); D.M. Glover; F.G. Grosveld (Frank); I. Dodd; J.H. Robinson

    1985-01-01

    textabstractWe have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libra

  10. Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

    DEFF Research Database (Denmark)

    Christensen, C B; Jørgensen, L; Jensen, A T

    2000-01-01

    Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L...

  11. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and aminoacid homology with the yeast DNA repair gene RAD10.

    NARCIS (Netherlands)

    M. van Duin (Mark); J. de Wit (Jan); H. Odijk (Hanny); A. Westerveld (Andries); A. Yasui (Akira); M.H.M. Koken (Marcel); J.H.J. Hoeijmakers (Jan); D. Bootsma (Dirk)

    1986-01-01

    textabstractThe human excision repair gene ERCC-7 was cloned after DNA mediated gene transfer to the CHO mutant 43-38, which is sensitive to ultraviolet light and mitomycin-C. We describe the cloning and sequence analysis of the ERCC-7 cDNA and partial characterization of the gene. ERCC.1 has a size

  12. Molecular cloning, functional expression, and tissue distribution of a novel human gap junction-forming protein, connexin-31.9. Interaction with zona occludens protein-1

    NARCIS (Netherlands)

    Nielsen, Peter A; Beahm, Derek L; Giepmans, Ben N G; Baruch, Amos; Hall, James E; Kumar, Nalin M

    2002-01-01

    A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.

  13. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

    Directory of Open Access Journals (Sweden)

    Hiroshi Kondo

    2015-01-01

    Full Text Available Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12 were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC, an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5, an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1 expression levels were enhanced. After treatment with dexamethasone (DEX, 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX, and keratinocyte growth factor (KGF, surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation.

  14. Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

    Science.gov (United States)

    Zhang, Yihua; Shen, Wenzheng; Sun, Bingjie; Lv, Changrong; Dou, Zhongying

    2011-02-01

    Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

  15. IN VITRO STUDY ON THE CLONING AND TRANSDUCTION OF HUMAN O6-METHYLGUANINE-DNA-METHYLTRANSFERASE CDNA INTO HUMAN UMBILICAL CORD BLOOD CD34+ CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O6-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT-PCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8′ 105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT-cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgene cord blood CD34+ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34+ cells and its expression provided an experimental foundation for gene therapy in clinical trial.

  16. Just Say No to NOTA: Why the Prohibition of Compensation for Human Transplant Organs in NOTA Should Be Repealed and a Regulated Market for Cadaver Organs Instituted.

    Science.gov (United States)

    Williams, Kristy Lynn; Finley, Marisa; Rohack, J James

    2014-01-01

    The United States faces a shortage of organs for transplantation; thousands of individuals die each year while waiting for organs. The organ donation system relies on altruism because the National Organ Transplantation Act (NOTA) prohibits donors from receiving valuable consideration for organs to be used for transplantation. This paper contains a proposal for a regulated market for cadaveric organs as a mechanism to increase the number of organs available for transplantation. A regulated market for cadaveric organs is appropriate in the United States for the following reasons: (1) it is consistent with the numerous ways in which bodies. are currently treated as commodities; (2) it is unlikely to further disadvantage the poor; (3) it would not expose organ recipients to undue harm; (4) in the current organ transplantation system, everyone except the organ donor, benefits financially; (5) the prohibition in NOTA is inconsistent with laws permitting next of kin to recover for damage to the body of a deceased family member; (6) the protection of the dignitary interests of organs for donation is inconsistent with the current protections of the dignitary interests of human specimens; and (7) permitting a market for organs promotes the American values of autonomy and liberty.

  17. Genomic organization and promoter cloning of the human X11α gene APBA1.

    LENUS (Irish Health Repository)

    Chai, Ka-Ho

    2012-05-01

    X11α is a brain specific multi-modular protein that interacts with the Alzheimer\\'s disease amyloid precursor protein (APP). Aggregation of amyloid-β peptide (Aβ), an APP cleavage product, is believed to be central to the pathogenesis of Alzheimer\\'s disease. Recently, overexpression of X11α has been shown to reduce Aβ generation and to ameliorate memory deficit in a transgenic mouse model of Alzheimer\\'s disease. Therefore, manipulating the expression level of X11α may provide a novel route for the treatment of Alzheimer\\'s disease. Human X11α is encoded by the gene APBA1. As evidence suggests that X11α expression can be regulated at transcription level, we have determined the gene structure and cloned the promoter of APBA1. APBA1 spans over 244 kb on chromosome 9 and is composed of 13 exons and has multiple transcription start sites. A putative APBA1 promoter has been identified upstream of exon 1 and functional analysis revealed that this is highly active in neurons. By deletion analysis, the minimal promoter was found to be located between -224 and +14, a GC-rich region that contains a functional Sp3 binding site. In neurons, overexpression of Sp3 stimulates the APBA1 promoter while an Sp3 inhibitor suppresses the promoter activity. Moreover, inhibition of Sp3 reduces endogenous X11α expression and promotes the generation of Aβ. Our findings reveal that Sp3 play an essential role in APBA1 transcription.

  18. Infectivity of chimeric human T-cell leukemia virus type I molecular clones assessed by naked DNA inoculation.

    Science.gov (United States)

    Zhao, T M; Robinson, M A; Bowers, F S; Kindt, T J

    1996-06-25

    Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.

  19. Cloning of Human Uroplakin Ⅱ Gene from Chinese Transitional Cell Carcinoma of Bladder and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To clone Uroplakin Ⅱ gene from Chinese transitional cell carcinoma (TCC) of bladder and construct its eukaryotic expression vector, the molecular cloning method was used to extract total RNA from a GⅢ/ T3N0M0 tissue sample of the bladder TCC patients. The primers were designed by Primer 5.0 software. Full length cDNA of Uroplakin Ⅱ gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), assayed by nucleic acid sequencing and then inserted between Xba Ⅰ and HindⅢ restrictive sites of eukaryotic expression vector pcDNA3.0. The recombinant was assayed by restricted enzyme digestion. Under the induction of Lipofectamine 2000, the recombinant was transfected into Uroplakin Ⅱ negative bladder cancer cell line EJ. Cellular expression levels of Uroplakin Ⅱ were detected by RT-PCR. The nucleic acid sequencing results indicated that Chinese Uroplakin Ⅱ cDNA (555 bp) was successfully cloned. The BLAST analysis demonstrated that the cloned sequence is 100 % homologous with sequences reported overseas. The GenBank accession number AY455312 was also registered. The results of restricted enzyme digestion indicated that eukaryotic vector pcDNA-UP Ⅱ for Uroplakin Ⅱ was successfully constructed.After being transferred with pcDNA-UPⅡ for 72 h, cellular Uroplakin Ⅱ mRNA levels were significantly improved (P<0.01). It is concluded that human Uroplakin Ⅱ gene was successfully cloned from Chinese TCC tissues, which provided a basis for further exploration of the roles of Uroplakin Ⅱ gene in TCC biological behaviors and potential strategies for targeted biological therapy of TCC.

  20. Identification of peptides from human pathogens able to cross-activate an HIV-1-gag-specific CD4+ T cell clone.

    Science.gov (United States)

    Venturini, Sara; Allicotti, Gina; Zhao, Yindong; Simon, Richard; Burton, Dennis R; Pinilla, Clemencia; Poignard, Pascal

    2006-01-01

    Antigen recognition by T cells is degenerate both at the MHC and the TCR level. In this study, we analyzed the cross-reactivity of a human HIV-1 gag p24-specific CD4(+) T cell clone obtained from an HIV-1-seronegative donor using a positional scanning synthetic combinatorial peptide library (PS-SCL)-based biometrical analysis. A number of decapeptides able to activate the HIV-1 gag-specific clone were identified and shown to correspond to sequences found in other human pathogens. Two of these peptides activated the T cell clone with the same stimulatory potency as the original HIV-1 gag p24 peptide. These findings show that an HIV-1-specific human T helper clone can react efficiently with peptides from other pathogens and suggest that cellular immune responses identified as being specific for one human pathogen (HIV-1) could arise from exposure to other pathogens.

  1. Clone-specific expression, transcriptional regulation, and action of interleukin-6 in human colon carcinoma cells

    Directory of Open Access Journals (Sweden)

    Fabjani Gerhild

    2008-01-01

    Full Text Available Abstract Background Many cancer cells produce interleukin-6 (IL-6, a cytokine that plays a role in growth stimulation, metastasis, and angiogenesis of secondary tumours in a variety of malignancies, including colorectal cancer. Effectiveness of IL-6 in this respect may depend on the quantity of basal and inducible IL-6 expressed as the tumour progresses through stages of malignancy. We therefore have evaluated the effect of IL-6 modulators, i.e. IL-1β, prostaglandin E2, 17β-estradiol, and 1,25-dihydroxyvitamin D3, on expression and synthesis of the cytokine at different stages of tumour progression. Methods We utilized cultures of the human colon carcinoma cell clones Caco-2/AQ, COGA-1A and COGA-13, all of which expressed differentiation and proliferation markers typical of distinct stages of tumour progression. IL-6 mRNA and protein levels were assayed by RT-PCR and ELISA, respectively. DNA sequencing was utilized to detect polymorphisms in the IL-6 gene promoter. Results IL-6 mRNA and protein concentrations were low in well and moderately differentiated Caco-2/AQ and COGA-1A cells, but were high in poorly differentiated COGA-13 cells. Addition of IL-1β (5 ng/ml to a COGA-13 culture raised IL-6 production approximately thousandfold via a prostaglandin-independent mechanism. Addition of 17β-estradiol (10-7 M reduced basal IL-6 production by one-third, but IL-1β-inducible IL-6 was unaffected. Search for polymorphisms in the IL-6 promoter revealed the presence of a single haplotype, i.e., -597A/-572G/-174C, in COGA-13 cells, which is associated with a high degree of transcriptional activity of the IL-6 gene. IL-6 blocked differentiation only in Caco-2/AQ cells and stimulated mitosis through up-regulation of c-myc proto-oncogene expression. These effects were inhibited by 10-8 M 1,25-dihydroxyvitamin D3. Conclusion In human colon carcinoma cells derived from well and moderately differentiated tumours, IL-6 expression is low and only marginally

  2. Human granulocyte colony stimulating factor (hG-CSF: cloning, overexpression, purification and characterization

    Directory of Open Access Journals (Sweden)

    Vanz Ana LS

    2008-04-01

    Full Text Available Abstract Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3 host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4% to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost

  3. Variations and voids: the regulation of human cloning around the world.

    Science.gov (United States)

    Pattinson, Shaun D; Caulfield, Timothy

    2004-12-13

    No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1). We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC) and non-reproductive cloning (NRC). While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.

  4. Variations and voids: the regulation of human cloning around the world

    Directory of Open Access Journals (Sweden)

    Caulfield Timothy

    2004-12-01

    Full Text Available Abstract Background No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality. Methods Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1. We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC and non-reproductive cloning (NRC. Results While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries. Conclusions There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.

  5. Fluorescent in-situ hybridization of cattle and sheep chromosomes with cloned human fragile-X DNA

    DEFF Research Database (Denmark)

    Ali, Ahmd; Thomsen, Preben Dybdahl; Babar, M.E.

    2009-01-01

    An extensive study on spontaneous and 5-Fluorodeoxyuridine induced fragile sites identified Xq31 in cattle (Bos taurus) and (Xq24, Xq26) in sheep (Ovis aries) in addition to several autosomal fragile sites (under publication). A ZOO-FISH study using three cloned human fragile-X probes with CCG....../CGG(n) trinucleotide repeat sequence was carried out to determine homology between human and bovine fragile-X. The hybridisation results showed only a weak signal on a human chromosome that was not an X with all three fragile site probes. No signals were detected in sheep chromosomes. The signal of all three human...... showed no signals whatsoever. It was therefore concluded that no homology existed between human and bovine fragile-X....

  6. Molecular cloning, characterization and expression of the heat shock protein 60 gene from the human pathogenic fungus Paracoccidioides brasiliensis.

    Science.gov (United States)

    Izacc, S M; Gomez, F J; Jesuino, R S; Fonseca, C A; Felipe, M S; Deepe, G S; Soares, C M

    2001-10-01

    A gene encoding the heat shock protein (HSP) 60 from Paracoccidioides brasiliensis (Pb) was cloned and characterized. The hsp60 gene is composed of three exons divided by two introns. Structural analysis of the promoter detected canonical sequences characteristic of regulatory regions from eukaryotic genes. The deduced amino acid sequence of the Pb hsp60 gene and the respective cloned cDNA consists of 592 residues highly homologous to other fungal HSP60 proteins. The hsp60 gene is present as a single copy in the genome, as shown by Southern blot analysis. The HSP60 protein was isolated from Pb yeast cellular extracts. N-terminal amino acid sequencing of HSP60 confirmed that the cloned hsp60 gene correlated to the predicted protein in Pb. HSP60 expression appeared to be regulated during form transition in Pb, as different levels of expression were detected in in vitro labeling of cells and northern blot analysis. The complete coding region of Pb hsp60 was fused with plasmid pGEX-4T-3 and expressed in Escherichia coli as a glutathione S-transferase-tagged recombinant protein. The protein reacted with a mouse monoclonal antibody raised to a human recombinant HSP60. Western immunoblot experiments demonstrated that the recombinant protein and the native HSP60 were recognized by sera from humans with paracoccidioidomycosis (PCM).

  7. Full-length clone and characterization of a human immunodeficiency virus type 1 subtype B' isolated from Hubei Province, China

    Institute of Scientific and Technical Information of China (English)

    TAN Jian-xin; KANG Xian-jiang; ZHANG Wei; LIU Ping-ping; TONG Xiao; YANG Rong-ge

    2007-01-01

    @@ There are two types of Human Immunodeficiency Virus (HIV): HIV-1 and HIV-2. HIV-1 dominates epidemics in many different parts of the world, and HIV-2 is principally responsible for the epidemic in western Africa. In China, Zeng et al1 have reported the first individual infected with HIV-1 in 1985. And in the 1990s,there was a severe epidemic involving the HIV-1 B' strain among people who sold blood and plasma in Henan,Hubei and adjacent provinces.2 To further study in HIV/AIDS vaccines and HIV-1 drug resistance for people in these regions, we need to construct an infectious HIV-1 B' molecular clone which is representative of the virus in these areas.3 To this end, we have isolated a HIV-1 B' virus from a child who was infected with HIV-1 from his mother in Hubei province, and have constructed a full-length clone from this genome.

  8. High level expression of bioactive recombinant human growth hormone in the milk of a cloned transgenic cow.

    Science.gov (United States)

    Salamone, Daniel; Barañao, Lino; Santos, Claudio; Bussmann, Leonardo; Artuso, Jorge; Werning, Carlos; Prync, Aida; Carbonetto, Cesar; Dabsys, Susana; Munar, Carlos; Salaberry, Roberto; Berra, Guillermo; Berra, Ignacio; Fernández, Nahuel; Papouchado, Mariana; Foti, Marcelo; Judewicz, Norberto; Mujica, Ignacio; Muñoz, Luciana; Alvarez, Silvina Fenández; González, Eliseo; Zimmermann, Juan; Criscuolo, Marcelo; Melo, Carlos

    2006-07-13

    Transgenic farm animals have been proposed as an alternative to current bioreactors for large scale production of biopharmaceuticals. However, the efficiency of both methods in the production of the same protein has not yet been established. Here we report the production of recombinant human growth hormone (hGH) in the milk of a cloned transgenic cow at levels of up to 5 g l(-1). The hormone is identical to that currently produced by expression in E. coli. In addition, the hematological and somatometric parameters of the cloned transgenic cow are within the normal range for the breed and it is fertile and capable of producing normal offspring. These results demonstrate that transgenic cattle can be used as a cost-effective alternative for the production of this hormone.

  9. Cloning, Eukaryotic Expression of Human ISG20 and Preliminary Study on the Effect of Its Anti-HBV

    Institute of Scientific and Technical Information of China (English)

    Youhua HAO; Dongliang YANG

    2008-01-01

    Human ISG20 gene was cloned and the effect of its anti-HBV was primarily studied. The ISG20 gene was amplified from HeLa cells by RT-PCR and recombinant vector expressing ISG20 was constructed by genetic engineering. The overexpression of ISG20 in HepG2 cells was detected by Western blot and the levels of secretion of HBs antigen and HBe antigen tested by ELISA. The results showed that: (1) Sequence of ISG20 cloned was consistent to that published in Genebank; (2) Recombinant vector expressing ISG20 could be expressed in HepG2 cells by transfection; (3) The overexpression of ISG20 protein could reduce the levels of the secretion of HBs antigen and HBe an-tigen in transfected HepG2 cells. It was suggested that the overexpression of recombinant ISG20 in culture cells could reduce the synthesis of HBV proteins.

  10. Cloning and Characterization of Multiple RNA Splicing Variants of LDH-C Gene in Human and Rat

    Directory of Open Access Journals (Sweden)

    Qinglian Zhang

    2013-06-01

    Full Text Available The expression of LDH-C (Lactate dehydrogenase C gene is restricted in mature germ cells; however multiple splice variants of LDH-C expressed in human cancers and yak normal testes were reported recently. In order to know if there are any LDH-C splice variants in human normal testes, we set out to clone the putative variants in human and rat. Four splicing variants in human testes, 1 splicing variant in human spermatozoa, 6 splicing variants in rat testes and 1 splicing variant in rat non-testes tissues (liver, heart and muscle were cloned. The putative polypeptides encoded by these variants were compared with the full-length LDH-C protein, the results showed that these putative polypeptides were truncated LDH-C proteins or truncated LDH-C proteins with a few amino acid residues different at N or C terminal. This suggested that these variants are possibly not used for translation, but targets of nonsense-mediated mRNA decay. Western blotting did not detect any bands with similar molecular weight as the putative polypeptides. RT-PCR showed that the expression levels of the splicing variants were significant during development of rat testes. The results indicate that LDH-C was not silenced by transcriptional repression in non-mature germ cells, but significantly transcripted and alternatively spliced.

  11. My Research Is Militants Most Secrecy. Human Killing Death Body Hide and Militants Exceptional Clone System.

    OpenAIRE

    Mohammad Abdul Khaleak

    2016-01-01

    : I know about the top most secret matter of the militants. All militants are exceptional clone. The process of cloning will never be disclosed by the militants. Jinn/Fairy is one kind of species. I have seen the Jinn/Fairy many times. Some men know. Some men have seen the Jinn/Fairy. Jinn is male and fairy is female. Jinn/Fairy can take shape of any man. Then can’t see in day light, do not have any teeth in mouth, no nails hands and legs fingers. Then in the thumb there is one bone in phalan...

  12. Molecular cloning and chromosomal assignment of the human brain-type phosphodiesterase I/nucleotide pyrophosphatase gene (PDNP2)

    Energy Technology Data Exchange (ETDEWEB)

    Kawagoe, Hiroyuki; Soma, Osamu; Goji, Junko [Kobe Univ. School of Medicine (Japan)] [and others

    1995-11-20

    Phosphodiesterase I/nucleotide pyrophosphatase is a widely expressed membrane-bound enzyme that cleaves diester bonds of a variety of substrates. We have cloned brain-type cDNA for this enzyme from rat brain and designated it PD-I{alpha}. In this study we have isolated cDNA and genomic DNA encoding human PD-I{alpha}. Human PD-I{alpha} cDNA, designated PDNP2 in HGMW nomenclature, has a 2589-nucleotide open reading frame encoding a polypeptide of 863 amino acids with a calculated M{sub r} of 99,034. Northern blot analysis revealed that human PD-I{alpha} transcript was present in brain, lung, placenta, and kidney. The database analysis showed that human PD-I{alpha} was identical with human autotaxin (ATX), a novel tumor motility-stimulating factor, except that human PD-I{alpha} lacks 156 nucleotides and 52 amino acids of human ATX. Human PD-I{alpha} and human ATX are likely to be alternative splicing products from the same gene. The 5{prime} region of the human PDNP2 gene contains four putative binding sites of transcription factor Sp1 without typical TATA or CAAT boxes, and there is a potential octamer binding motif in intron 2. From the results of fluorescence in situ hybridization, the human PDNP2 gene is located at chromosome 8q24.1. 17 refs., 3 figs.

  13. Cloning of Human α-defensin-1 (HNP-1) Gene and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    Hua-Hua CHEN; Jing-Ping Ou YANG; Bao-Hua WANG; Yue Yang; Han-Qiao ZHENG

    2005-01-01

    @@ 1 Introduction Defensins are small cationic antimicrobial peptides that function in the host's innate immune system. The human defensin family includes three small peptides from the azurophil granules of polymorphonuclear cells named human neutrophil peptide (HNP)-1, HNP-2, HNP-3,which consist 5%-7% of the protein of human neutrophil. HNP-4 is approximately one hundred times less abundant. They demonstrate antibacterial, antifungal and antiviral properties in vitro. HNPs are important component of nonoxidative mechanism in macrophages, and can direct inactivate the enveloped viruses. Because only special cells express defensins. And it is hard to extract them naturally and the production is few. So researcher expect to obtain defensins highly through heterogenous expression by gene engineering technology. In order to express HNP-1, we cloned the cDNA of HNP-1 from human polymorphonuclear cells in peripheral blood, and constructed its eukaryotic expression vector, which provided a base for the further study on its mechanism of antimicrobial effect.

  14. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

    DEFF Research Database (Denmark)

    Wewer, U M; Gerecke, D R; Durkin, M E

    1994-01-01

    Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human...

  15. Molecular cloning of a new angiopoietinlike factor from the human cornea

    NARCIS (Netherlands)

    Peek, R; van Gelderen, BE; Bruinenberg, M; Kijlstra, A

    PURPOSE. To isolate tissue-specific gene products that contribute to corneal integrity. METHODS. A cDNA library was constructed and differentially hybridized. Cornea-specific clones were purified and further characterized. RESULTS. In this study cornea-specific gene products were isolated by

  16. cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

    Institute of Scientific and Technical Information of China (English)

    QI Zhen-yu; HUI Guo-zhen; LI Yao; ZHOU Zong-xiang; GU Shao-hua; YING Kang; XIE Yi

    2005-01-01

    Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5'RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b).Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.

  17. Pluripotent embryonal carcinoma clones derived from the human teratocarcinoma cell line Tera-2. Differentiation in vivo and in vitro.

    Science.gov (United States)

    Andrews, P W; Damjanov, I; Simon, D; Banting, G S; Carlin, C; Dracopoli, N C; Føgh, J

    1984-02-01

    We have derived and characterized single cell clones from a xenograft tumor of the teratocarcinoma cell line Tera-2. Isozyme and chromosomal analyses confirmed their common origin. When cultures of the clones were maintained at a high cell density, many cells exhibited a morphology and cell surface antigen phenotype typical of human embryonal carcinoma cells. These features included a high nucleo-cytoplasmic ratio, prominent nucleoli, and the expression of the globoseries glycolipid antigen SSEA-3. In addition, other cells, in many respects resembling these typical embryonal carcinoma cells, were distinguished by a marked tendency to accumulate cytoplasmic glycogen. Similar cells, together with more differentiated cells, were seen in low passage cultures of Tera-2 itself. When the clones were grown at a low cell density many cells assumed a larger, flatter shape, a few with multiple nucleoli. Also, the fucosylated lactosamine antigen SSEA-1 appeared on some cells, whereas expression of SSEA-3 and HLA-A,B,C tended to be reduced. Often the synthesis of fibronectin was increased. However, no obvious cytoplasmic differentiation was seen upon ultrastructural examination, and synthesis of human chorionic gonadotropin, alpha-fetoprotein, and laminin was not detected. In contrast to the limited spontaneous changes seen in culture, marked differentiation occurred in tumors obtained following injection of the cells into athymic (nu/nu) mice. In additional to embryonal carcinoma cells, these tumors contained a variety of somatic tissues that included glandular structures, possibly related to the primitive gut, and neural elements. These cell lines derived from Tera-2 constitute the first example of clonal human embryonal carcinoma cells, adapted to growth in vitro, that have retained the capacity for differentiation into diverse somatic tissues.

  18. Comparative mapping of a gorilla-derived alpha satellite DNA clone on great ape and human chromosomes.

    Science.gov (United States)

    Baldini, A; Miller, D A; Shridhar, V; Rocchi, M; Miller, O J; Ward, D C

    1991-11-01

    We have isolated an alpha satellite DNA clone, pG3.9, from gorilla DNA. Fluorescence in situ hybridization on banded chromosomes under high stringency conditions revealed that pG3.9 identifies homologous sequences at the centromeric region of ten gorilla chromosomes, and, with few exceptions, also recognizes the homologous chromosomes in human. A pG3.9-like alphoid DNA is present on a larger number of orangutan chromosomes, but, in contrast, is present on only two chromosomes in the chimpanzee. These results show that the chromosomal subsets of related alpha satellite DNA sequences may undergo different patterns of evolution.

  19. Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

    Energy Technology Data Exchange (ETDEWEB)

    Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

    1987-12-01

    Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

  20. Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, M.

    1986-06-01

    By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.

  1. Transgenic mammalian species, generated by somatic cell cloning, in biomedicine, biopharmaceutical industry and human nutrition/dietetics--recent achievements.

    Science.gov (United States)

    Samiec, M; Skrzyszowska, M

    2011-01-01

    Somatic cell cloning technology in mammals promotes the multiplication of productively-valuable genetically engineered individuals, and consequently allows also for standardization of transgenic farm animal-derived products, which, in the context of market requirements, will have growing significance. Gene farming is one of the most promising areas in modern biotechnology. The use of live bioreactors for the expression of human genes in the lactating mammary gland of transgenic animals seems to be the most cost-effective method for the production/processing of valuable recombinant therapeutic proteins. Among the transgenic farm livestock species used so far, cattle, goats, sheep, pigs and rabbits are useful candidates for the expression of tens to hundreds of grams of genetically-engineered proteins or xenogeneic biopreparations in the milk. At the beginning of the new millennium, a revolution in the treatment of disease is taking shape due to the emergence of new therapies based on recombinant human proteins. The ever-growing demand for such pharmaceutical or nutriceutical proteins is an important driving force for the development of safe and large-scale production platforms. The aim of this paper is to present an overall survey of the state of the art in investigations which provide the current knowledge for deciphering the possibilities of practical application of the transgenic mammalian species generated by somatic cell cloning in biomedicine, the biopharmaceutical industry, human nutrition/dietetics and agriculture.

  2. What is Cloning?

    Science.gov (United States)

    Donate Home Cloning What is Cloning What is Cloning Clones are organisms that are exact genetic copies. ... clones made through modern cloning technologies. How Is Cloning Done? Many people first heard of cloning when ...

  3. Cloning, structural characterization, and chromosomal localization of the gene encoding the human prostaglandin E(2) receptor EP2 subtype.

    Science.gov (United States)

    Smock, S L; Pan, L C; Castleberry, T A; Lu, B; Mather, R J; Owen, T A

    1999-09-17

    Northern blot analysis of human placental RNA using a probe to the 5' end of the human prostaglandin E(2) (PGE(2)) EP2 receptor subtype coding region revealed the existence of a high abundance, low molecular weight transcript. To investigate the origin of this transcript, and its possible relationship to the human EP2 mRNA, we have cloned and characterized the gene encoding the human PGE(2) EP2 receptor subtype, identified transcriptional initiation and termination sites in two tissues (spleen and thymus), and determined its chromosomal localization. The human EP2 gene consists of two exons separated by a large intron, utilizes a common initiation site in both spleen and thymus at 1113 bp upstream of the translation initiation site, and has 3' transcript termini at 1140 bp and 1149 bp downstream of the translation stop site in spleen and thymus respectively. Southern and fluorescence in situ hybridization analysis demonstrated the human EP2 gene to be a single copy gene located in band 22 of the long arm of chromosome 14 (14q22). Though our initial interest in this gene was to investigate potential differential splicing of the human EP2 gene in placenta, this work demonstrates that the atypical transcript observed in placenta probably arises from a distinct, yet related, gene. Knowledge of the sequence, structure, and transcription events associated with the human EP2 gene will enable a broader understanding of its regulation and potential role in normal physiology and disease.

  4. The Dolly case, the Polly drug, and the morality of human cloning.

    Science.gov (United States)

    Schramm, F R

    1999-01-01

    The year 1996 witnessed the cloning of the lamb Dolly, based on the revolutionary somatic cell nuclear transfer (SCNT) technique, developed by researchers from the Roslin Institute in Edinburgh, Scotland. This fact marked a relevant biotechnoscientific innovation, with probable significant consequences in the field of public health, since in principle it allows for expanding possibilities for the reproductive autonomy of infertile couples and carriers of diseases of mitochondrial origin. This article expounds on 1) the experiment's technical data and the theoretical implications for the biological sciences; 2) the public's perception thereof and the main international documents aimed at the legal and moral regulation of the technique; and 3) the moral arguments for and against cloning, from the point of view of consequentialist moral theory. We conclude that in the current stage of the debate on the morality of cloning, in which there are no cogent deontological arguments either for or against, weighing the probability of risks and benefits is the only reasonable way of dealing with the issue in societies that consider themselves democratic, pluralistic, and tolerant.

  5. The Dolly case, the Polly drug, and the morality of human cloning

    Directory of Open Access Journals (Sweden)

    Schramm Fermin Roland

    1999-01-01

    Full Text Available The year 1996 witnessed the cloning of the lamb Dolly, based on the revolutionary somatic cell nuclear transfer (SCNT technique, developed by researchers from the Roslin Institute in Edinburgh, Scotland. This fact marked a relevant biotechnoscientific innovation, with probable significant consequences in the field of public health, since in principle it allows for expanding possibilities for the reproductive autonomy of infertile couples and carriers of diseases of mitochondrial origin. This article expounds on 1 the experiment's technical data and the theoretical implications for the biological sciences; 2 the public's perception thereof and the main international documents aimed at the legal and moral regulation of the technique; and 3 the moral arguments for and against cloning, from the point of view of consequentialist moral theory. We conclude that in the current stage of the debate on the morality of cloning, in which there are no cogent deontological arguments either for or against, weighing the probability of risks and benefits is the only reasonable way of dealing with the issue in societies that consider themselves democratic, pluralistic, and tolerant

  6. Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene

    Institute of Scientific and Technical Information of China (English)

    Zhi-Ming Hap; Jin-Yan Luo; Jin Cheng; Quan-Yin Wang; Guang-Xiao Yang

    2003-01-01

    AIM: To design a hammerhead ribozyme targeting humantelomerase reverse transcriptase (hTERT) and clone it's genefor future use in the study of tumor gene therapy.METHODS: Using the software RNAstructure, the secondarystructure of hTERT mRNA was predicted and the cleavagesite of ribozyme was selected. A hammerhead ribozymetargeting this site was designed and bimolecular fold betweenthe ribozyme and hTERT was predicted. The DNA encodingthe ribozyme was synthesized and cloned into pGEMEX-1and the sequence of the ribozyme gene was confirmed byDNA sequencing.RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosenas the cleavage site of the ribozyme. The designed ribozymewas comprised of 22nt catalytic core and 17nt flankingsequence. Computer-aided prediction suggested that theribozyme and hTERT mRNA could cofold into a properconformation. Endonuclease restriction and DNA sequencingconfirmed the correct insertion of the ribozyme gene intothe vector pGEMEX-1.CONCLUSION: This fundamental work of successfuldesigning and cloning of an anti-hTERT hammerheadribozyme has paved the way for further study of inhibitingtumor cell growth by cleaving hTERT mRNA with ribozyme.

  7. Recombinant human albumin supports single cell cloning of CHO cells in chemically defined media.

    Science.gov (United States)

    Zhu, Jiang; Wooh, Jong Wei; Hou, Jeff Jia Cheng; Hughes, Benjamin S; Gray, Peter P; Munro, Trent P

    2012-01-01

    Biologic drugs, such as monoclonal antibodies, are commonly made using mammalian cells in culture. The cell lines used for manufacturing should ideally be clonal, meaning derived from a single cell, which represents a technically challenging process. Fetal bovine serum is often used to support low cell density cultures, however, from a regulatory perspective, it is preferable to avoid animal-derived components to increase process consistency and reduce the risk of contamination from adventitious agents. Chinese hamster ovary (CHO) cells are the most widely used cell line in industry and a large number of serum-free, protein-free, and fully chemically defined growth media are commercially available, although these media alone do not readily support efficient single cell cloning. In this work, we have developed a simple, fully defined, single-cell cloning media, specifically for CHO cells, using commercially available reagents. Our results show that a 1:1 mixture of CD-CHO™ and DMEM/F12 supplemented with 1.5 g/L of recombinant albumin (Albucult®) supports single cell cloning. This formulation can support recovery of single cells in 43% of cultures compared to 62% in the presence of serum.

  8. Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

    Institute of Scientific and Technical Information of China (English)

    Ge-Jian Zhu; Ying-Nian Yu; Xin Li; Yu-Li Qian

    2002-01-01

    AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 ( CYP2 C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2 C9 eDNA was cloned and expressed stably in CHL cellsMETHODS: After extraction of total RNA from human livertissue, the human CYP2C9 eDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR),and cloned into cloning vector pGEM-T. The cDNA fragmentwas identified by DNA sequencing and subcloned into amammalian expression vector pREP9. A transgenic cell linewas established by transfecting the recombinant vector ofpREP9-CYP2C9 into CHL cells. The enzyme activity ofCYP2C9 catalyzing oxidation of tolbutamide to hydroxytolbutamide in S9 fraction of the cell was determined by highperformance liquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from theeDNA segment was identical to that of CYP2 C9 * 1, the wildtype CYP2 C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acidsequence was the same, L7, P382. The S9 fraction of theestablished cell line metabolizes tolbutamide to hydroxytolbutamide; tolbutamide hydroxylass activity was found to be0.465 ± 0.109 μmol@ min-1 . g1 S9 protein or 8.62 ± 2.02 mol@ min 1 ~mol-1 CYP, but was undetectable in parental CHL cell.CONCLUSION: The cDNA of human CYP2C9 was successfullycloned and a cell line of CHL- CYP2C9, efficiently expressingthe protein of CYP2C9, was established.

  9. Activation of resting human B cells by helper T-cell clone supernatant: characterization of a human B-cell-activating factor.

    Science.gov (United States)

    Diu, A; Gougeon, M L; Moreau, J L; Reinherz, E L; Thèze, J

    1987-12-01

    The effects of helper T-cell clone supernatants on resting human B cells were investigated. Four different helper T-cell clones (two T4+ and two T8+) were stimulated by anti-T3 monoclonal antibodies on Sepharose beads or anti-T11(2) plus anti-T11(3) monoclonal antibodies. The supernatants from these activated clones induced the proliferation of highly purified resting B lymphocytes from the peripheral blood. The B cells exhibited a cell size and a surface-antigen pattern (4F2 antigen and transferrin receptor) of phase G0 B cells, and they were functionally resting. In response to T-cell supernatants a large fraction of the B cells enlarged and expressed 4F2 antigens and transferrin receptors. In gel filtration, the corresponding activity migrated with an apparent Mr of 12,000-15,000. Our findings strongly support the existence of a human B-cell-activating factor acting on resting B cells and causing them to enter phase G1 of the cell cycle.

  10. Cloning, expression and nuclear localization of human NPM3, a member of the nucleophosmin/nucleoplasmin family of nuclear chaperones

    Directory of Open Access Journals (Sweden)

    Ganguly Amit

    2001-11-01

    Full Text Available Abstract Background Studies suggest that the related proteins nucleoplasmin and nucleophosmin (also called B23, NO38 or numatrin are nuclear chaperones that mediate the assembly of nucleosomes and ribosomes, respectively, and that these activities are accomplished through the binding of basic proteins via their acidic domains. Recently discovered and less well characterized members of this family of acidic phosphoproteins include mouse nucleophosmin/nucleoplasmin 3 (Npm3 and Xenopus NO29. Here we report the cloning and initial characterization of the human ortholog of Npm3. Results Human genomic and cDNA clones of NPM3 were isolated and sequenced. NPM3 lies 5.5 kb upstream of FGF8 and thus maps to chromosome 10q24-26. In addition to amino acid similarities, NPM3 shares many physical characteristics with the nucleophosmin/nucleoplasmin family, including an acidic domain, multiple potential phosphorylation sites and a putative nuclear localization signal. Comparative analyses of 14 members of this family from various metazoans suggest that Xenopus NO29 is a candidate ortholog of human and mouse NPM3, and they further group both proteins closer with the nucleoplasmins than with the nucleophosmins. Northern blot analysis revealed that NPM3 was strongly expressed in all 16 human tissues examined, with especially robust expression in pancreas and testis; lung displayed the lowest level of expression. An analysis of subcellular fractions of NIH3T3 cells expressing epitope-tagged NPM3 revealed that NPM3 protein was localized solely in the nucleus. Conclusions Human NPM3 is an abundant and widely expressed protein with primarily nuclear localization. These biological activities, together with its physical relationship to the chaparones nucleoplasmin and nucleophosmin, are consistent with the proposed function of NPM3 as a molecular chaperone functioning in the nucleus.

  11. Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Lagasse, E.; Clerc, R.G.

    1988-06-01

    The cellular mechanisms involved in chronic inflammatory processes are poorly understood. This is especially true for the role of macrophages, which figure prominently in the inflammatory response. Two proteins, MRP8 and MRP14, which are expressed in infiltrate macrophages during inflammatory reactions but not in normal tissue macrophages, which have been characterized. Here the authors report that MRP8 and MRP14 mRNAs are specially expressed in human cells of myeloid origin and that their expression is regulated during monocycle-macrophage and granulocyte differentiation. To initiate the analysis of cis-acting elements governing the tissue-specific expression of the MRP genes, the authors cloned the human genes encoding MRP8 and MRP14. Both genes contain three exons, are single copy, and have a strikingly similar organization. They belong to a novel subfamily of highly homologous calcium-binding proteins which includes S100..cap alpha.., S100BETA, intestinal calcium-binding protein, P11, and calcyclin (2A9). A transient expression assay was devised to investigate the tissue-specific regulatory elements responsible for MRP gene expression after differentiation in leukemia HL60 cells. The results of this investigation demonstrated that the cis-acting element responsible for MRP expression are present on the cloned DNA fragment containing the MRP gene loci.

  12. Integrative Annotation of 21,037 Human Genes Validated by Full-Length cDNA Clones

    Energy Technology Data Exchange (ETDEWEB)

    Imanishi, Tadashi; Itoh, Takeshi; Suzuki, Yutaka; O' Donovan, Claire; Fukuchi, Satoshi; Koyanagi, Kanako O.; Barrero, Roberto A.; Tamura, Takuro; Yamaguchi-Kabata, Yumi; Tanino, Motohiko; Yura, Kei; Miyazaki, Satoru; Ikeo, Kazuho; Homma, Keiichi; Kasprzyk, Arek; Nishikawa, Tetsuo; Hirakawa, Mika; Thierry-Mieg, Jean; Thierry-Mieg, Danielle; Ashurst, Jennifer; Jia, Libin; Nakao, Mitsuteru; Thomas, Michael A.; Mulder, Nicola; Karavidopoulou, Youla; Jin, Lihua; Kim, Sangsoo; Yasuda, Tomohiro; Lenhard, Boris; Eveno, Eric; Suzuki, Yoshiyuki; Yamasaki, Chisato; Takeda, Jun-ichi; Gough, Craig; Hilton, Phillip; Fujii, Yasuyuki; Sakai, Hiroaki; Tanaka, Susumu; Amid, Clara; Bellgard, Matthew; de Fatima Bonaldo, Maria; Bono Hidemasa; Bromberg, Susan K.; Brookes, Anthony J.; Bruford, Elspeth; Carninci Piero; Chelala, Claude; Couillault, Christine; de Souza, Sandro J.; Debily, Marie-Anne; Devignes, Marie-Dominique; Dubchak, Inna; Endo, Toshinori; Estreicher, Anne; Eyras, Eduardo; Fukami-Kobayashi, Kaoru; Gopinath, Gopal R.; Graudens, Esther; Hahn, Yoonsoo; Han, Michael; Han, Ze-Guang; Hanada, Kousuke; Hanaoka, Hideki; Harada, Erimi; Hashimoto, Katsuyuki; Hinz, Ursula; Hirai, Momoki; Hishiki, Teruyoshi; Hopkinson, Ian; Imbeaud, Sandrine; Inoko, Hidetoshi; Kanapin, Alexander; Kaneko, Yayoi; Kasukawa, Takeya; Kelso, Janet; Kersey, Paul; Kikuno Reiko; Kimura, Kouichi; Korn, Bernhard; Kuryshev, Vladimir; Makalowska, Izabela; Makino Takashi; Mano, Shuhei; Mariage-Samson, Regine; Mashima, Jun; Matsuda, Hideo; Mewes, Hans-Werner; Minoshima, Shinsei; Nagai, Keiichi; Nagasaki, Hideki; Nagata, Naoki; Nigam, Rajni; Ogasawara, Osamu; Ohara, Osamu; Ohtsubo, Masafumi; Okada, Norihiro; Okido, Toshihisa; Oota, Satoshi; Ota, Motonori; Ota, Toshio; Otsuki, Tetsuji; Piatier-Tonneau, Dominique; Poustka, Annemarie; Ren, Shuang-Xi; Saitou, Naruya; Sakai, Katsunaga; Sakamoto, Shigetaka; Sakate, Ryuichi; Schupp, Ingo; Servant, Florence; Sherry, Stephen; Shiba Rie; et al.

    2004-01-15

    The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/). It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs), identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4 percent of the human genome sequence (National Center for Biotechnology Information build 34 assembly) may contain misassembled or missing regions. We found that 6.5 percent of the human gene candidates (1,377 loci) did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for nonprotein-coding RNA

  13. Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

    Directory of Open Access Journals (Sweden)

    Tadashi Imanishi

    2004-06-01

    Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

  14. Reproductive cloning, genetic engineering and the autonomy of the child: the moral agent and the open future.

    Science.gov (United States)

    Mameli, M

    2007-02-01

    Some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies would undermine the autonomy of the resulting child. In this paper, two versions of this view are discussed. According to the first version, the autonomy of cloned and genetically engineered people would be undermined because knowledge of the method by which these people have been conceived would make them unable to assume full responsibility for their actions. According to the second version, these biotechnologies would undermine autonomy by violating these people's right to an open future. There is no evidence to show that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions; there is also no evidence for the claim that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans.

  15. Analysis of allelic expression patterns of IL-2, IL-3, IL-4, and IL-13 in human CD4+ T cell clones.

    NARCIS (Netherlands)

    Bayley, J.P.; Bakker, AM; Kaijzel, EL; Wierenga, EA; Pouw Kraan, van der C.T.M.; Huizinga, T.W.; Verweij, C.L.

    2003-01-01

    The occurrence of monoallelic expression of cytokine genes in single cells has been convincingly demonstrated, but there have been few reports of this phenomenon in T cell clones. Here we describe studies on the expression of alleles of the human genes encoding IL-2, IL-3, IL-4, and IL-13 in human C

  16. Efficient cDNA cloning by direct phenotypic correction of a mutant human cell line (HPRT-) using an Epstein-Barr virus derived cDNA expression vector.

    NARCIS (Netherlands)

    P.B.G.M. Belt; W. Jongmans; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude); P. van de Putte (Pieter)

    1991-01-01

    textabstractHuman cells are, in general, poor recipients of foreign DNA, which has severely hampered the cloning of genes by direct phenotypic correction of deficient human cell lines after DNA mediated gene transfer. In this communication a methodology is presented which largely circumvents this pr

  17. Familial hypercholesterolemia and atherosclerosis in cloned minipigs created by DNA transposition of a human PCSK9 gain-of-function mutant

    DEFF Research Database (Denmark)

    Al-Mashhadi, Rozh H; Sørensen, Charlotte B; Kragh, Peter M

    2013-01-01

    dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low...

  18. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  19. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  20. Cloning, tissue expression pattern characterization and chromosome localization of human peptide methionine sulfoxide reductase cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Oxidation and reduction of some amino acids are one of the molecular mechanisms for regulating the function of proteins. The oxidation of methionine (Met) to methionine sulfoxide (Met(O)) results in decreasing or loss of the biological activity of related proteins. It was found that peptide methionine sulfoxide reductase (msrA) can reduce Met(O) to Met and therefore restored the biological function of the oxidized proteins. To reveal the methionine oxidation-reduction mechanism in human body, in this study, the cDNA sequence of bovine msrA was used as an information-probe to screen the human EST database. Based on a contig assembled from homologous ESTs, a 1 256-bp human MSRA cDNA was cloned from several human cDNA libraries. The cDNA contains an open reading frame (ORF) of 705 bp in length, which encodes 235 amino acid residues. Homology comparison revealed that human MSRA shares 88% and 61% identities with bovine and Escherichia coli msrA protein respectively. Expression pattern analysis revealed a single 1.6-kb transcript of human MSRA in most human tissues and with highest expression in kidney. By radiation hybrid panel mapping, the gene was localized to human chromosome 8p22-23 between markers D8S518 and D8S550. There are 2 human inherited diseases Keratolytic Winter Erythema and Microcephaly related genes in this region, it is inferred that human MSRA might be the candidate of the two diseases.

  1. [Cloning: necessary reflections on the imaginary].

    Science.gov (United States)

    Minahim, María Auxiliadora

    2009-01-01

    The article covers the innumerable reasons given for using cloning for therapeutic and reproductive purposes. The most commonly used argument in favour of the procedure has been that of preserving human dignity, which would include the wide exercising of personal autonomy without restrictions of an ethical nature. This view is countered by questions relating to the use of the technique, namely self-determination and the loss of the integrity of the species, which would include the transformation of a generation through the production of human beings and tissues. It must also be made clear that therapeutic cloning (which is carried out through the use of stem cells) is not yet a reality in the scientific world, with the result that the procedure that is supposedly necessary, which argues in favour of the destruction of the young embryo is misleading, as are also certain discourses used to refer to the theme and the science. Criminal law, on prohibiting this practice is anticipating it becoming a reality, protecting legal rights that affect supra-individual interests, such as the destruction of the young embryo, one of the issues of concern to ADIN (Acción Directa de Inconstitucionalidad en Brasil - Direct Action on Unconstitutionality in Brazil) 3510-0.

  2. Quantum cloning

    OpenAIRE

    Scarani, Valerio; Iblisdir, Sofyan; Gisin, Nicolas; Acin, Antonio

    2005-01-01

    The impossibility of perfectly copying (or cloning) an arbitrary quantum state is one of the basic rules governing the physics of quantum systems. The processes that perform the optimal approximate cloning have been found in many cases. These "quantum cloning machines" are important tools for studying a wide variety of tasks, e.g. state estimation and eavesdropping on quantum cryptography. This paper provides a comprehensive review of quantum cloning machines (both for discrete-dimensional an...

  3. Physiological level Production of Antigen-Specific Human Immunoglobulin in Cloned Transchromosomic Cattle

    OpenAIRE

    Akiko Sano; Hiroaki Matsushita; Hua Wu; Jin-An Jiao; Poothappillai Kasinathan; Eddie J. Sullivan; Zhongde Wang; Yoshimi Kuroiwa

    2013-01-01

    Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human...

  4. Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant Mrna transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

  5. Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

    Institute of Scientific and Technical Information of China (English)

    黄欣; 赵忠良; 袁正隆; 张明徽; 朱学军; 陈国友; 曹雪涛

    2000-01-01

    To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to- be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

  6. Cloning and characterization of a novel deletion mutant of heterogeneous nuclear ribonucleoprotein M4 from human dendritic cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To identify differentially expressed genes from antigen-stimulated human dendritic cells (DC), subtractive cloning was adopted and more than ten novel genes differentially expressed were cloned. One is a deletion mutant of heterogeneous nuclear ribonucleoprotein (hnRNP) M4 in which the residues from 159 to 197 of hnRNP M4 have been absent. The deletion mutant was shown to be co-expressed with hnRNP M4 in cell lines. The mutant was expressed in antigen-stimulated DC but not in normal DC. Northern blot analysis revealed the presence of a major hnRNP M4 deletion mutant mRNA transcript of 2.4 kilobase with the highest levels in peripheral lymphocytes, lung, liver and spleen. It was also expressed in bone marrow-derived stromal cells (BMSC), BMSC treated with several cytokines but not in BMSC treated with TNF-a. The results revealed a new member of hnRNP family and suggested that hnRNP would participate in antigen process and presentation.

  7. Prostitution, disability and prohibition

    DEFF Research Database (Denmark)

    Thomsen, Frej Klem

    2015-01-01

    Criminalisation of prostitution, and minority rights for disabled persons, are important contemporary political issues. The article examines their intersection by analysing the conditions and arguments for making a legal exception for disabled persons to a general prohibition against purchasing...... sexual services. It explores the badness of prostitution, focusing on and discussing the argument that prostitution harms prostitutes, considers forms of regulation and the arguments for and against with emphasis on a liberty-based objection to prohibition, and finally presents and analyses three...

  8. 40 CFR 26.203 - Prohibition of research conducted or supported by EPA involving intentional exposure of any human...

    Science.gov (United States)

    2010-07-01

    ... her fetus), a nursing woman, or child. 26.203 Section 26.203 Protection of Environment ENVIRONMENTAL... Involving Intentional Exposure of Human Subjects who are Children or Pregnant or Nursing Women § 26.203... is a pregnant woman (and therefore her fetus), a nursing woman, or child. Notwithstanding any other...

  9. Why Languages Prefer Prohibitives

    Institute of Scientific and Technical Information of China (English)

    Johan van der Auwera

    2006-01-01

    This paper deals with prohibitive markers, i.e., negative markers that are more or less dedicated to the expression of a prohibition. It documents the variety in the formal make-up of these markers and it confims the earlier claims that they are frequent everywhere, with at least one exception, viz., Western Europe. Four origins are discussed:prohibitive markers may derive from predicative constructions, they may appear as a side product of Jespersen's cycle,they may derive from a univerbation of imperative and negative markers, and they may be borrowed. As explanation is offered as to why languages prefer to have prohibitive markers. It is argued that attempts to explain this preference in terms of morphosyntax are misguided. Instead a frequency-based semantic explanation is offered. The most frequent use of negatives are declarative, thereby inviting a static ‘it is not the case that' paraphrase. It is important, however, to mark clearly that prohibitives are instances of a dynamic ‘let it be the case that' appeal. The paper ends on a discussion of languages that do not employ prohibitive markers.

  10. Human sulfotransferase SULT1C1: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Her, Chengtao; Weinshilboum, R.M. [Mayo Foundation, Rochester, MN (United States); Kaur, G.P. [Temple Univ. Medical School, Philadelphia, PA (United States)] [and others

    1997-05-01

    We have isolated and sequenced a cDNA that encodes an apparent human orthologue of a rat sulfotransferase (ST) cDNA that has been referred to as {open_quotes}ST1C1{close_quotes} - although it was recently recommended that sulfotransferase proteins and cDNAs be abbreviated {open_quotes}SULT.{close_quotes} The new human cDNA was cloned from a fetal liver-spleen cDNA library and had an 888-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 62% identical with that encoded by the rat ST1C1 cDNA and included signature sequences that are conserved in all cytosolic SULT enzymes. Dot blot analysis of mRNA from 50 human tissues indicated that the cDNA was expressed in adult human stomach, kidney, and thyroid, as well as fetal kidney and liver. Northern blot analyses demonstrated that the major SULT1C1 mRNA in those same tissues was 1.4 kb in length. We next determined the partial human SULT1C1 gene sequence for a portion of the 5{prime}-terminus of one intron. That sequence was used to design SULT1C1 gene-specific primers that were used to perform the PCR with DNA from human/rodent somatic cell hybrids to demonstrate that the gene was located on chromosome 2. PCR amplifications performed with human chromosome 2/rodent hybrid cell DNA as template sublocalized SULT1C1 to a region between bands 2q11.1 and 2q11.2. 14 refs., 2 figs.

  11. Cloning and Expression of Human Grn Gene%重组人Grn的克隆与表达

    Institute of Scientific and Technical Information of China (English)

    谌思; 粟艳林; 李悦; 彭小宁

    2015-01-01

    目的:构建人Grn基因原核表达载体并观察其表达。方法:从人单核细胞系THP-1细胞cDNA中扩增出Grn片段,通过酶切与连接,将Grn片段构建到pGEX-4T-2质粒载体上,再转化入感受态细胞TOP10,菌落PCR鉴定阳性克隆并测序分析,测序正确的重组质粒再转化入感受态细胞DE3中表达蛋白,用Western blot鉴定结果。结果:构建的pGEX-4T-2表达载体作PCR和双酶切鉴定,证实其中有目的片段完整插入,插入片段测序结果与Grn序列设计完全一致,将其转化入DE3中,Western blot结果表明在细菌裂解上清有分子质量为91KD的融合蛋白。结论:重组人Grn的克隆与表达成功,为进一步研究Grn的功能奠定了基础。%ObjectsTo construct a prokaryotic expression vector containing human Grn gene and observe its expression. MethodsThe gene Grn was amplifified by PCR from THP-1 cDNA. The cloned gene was digested by restrictive endonucle-ase and subcloned into eulcaryotic experssion vecter pGEX-4T-2. Grn had been cloned into pGEX-4T-2, and the recom-bined plasmid was transduced into TOP10. The positive cloning was identified by PCR and sequencing. The recombinant plasmid was transformed into competent cell of DE3. The expression of GRN was analyzed by Western blot.ResultsIt was verified by PCR and nucleotide sequencing that the constructed Grn eukaryotic vector was correct. After pGEX-4T-2 was transfected into DE3, a GST fusion protein molecule of91KD was found in supernatant of lysis of bactoria by Western blot. ConclusionA prokaryotic expression plasmid containing human grn gene is successfully constructed, and it can express out objectiveprotein, which has laid a concrete fundation for future study on grn.

  12. [Nuclear transfer and therapeutic cloning].

    Science.gov (United States)

    Xu, Xiao-Ming; Lei, An-Min; Hua, Jin-Lian; Dou, Zhong-Ying

    2005-03-01

    Nuclear transfer and therapeutic cloning have widespread and attractive prospects in animal agriculture and biomedical applications. We reviewed that the quality of oocytes and nuclear reprogramming of somatic donor cells were the main reasons of the common abnormalities in cloned animals and the low efficiency of cloning and showed the problems and outlets in therapeutic cloning, such as some basic problems in nuclear transfer affected clinical applications of therapeutic cloning. Study on isolation and culture of nuclear transfer embryonic stem (ntES) cells and specific differentiation of ntES cells into important functional cells should be emphasized and could enhance the efficiency. Adult stem cells could help to cure some great diseases, but could not replace therapeutic cloning. Ethics also impeded the development of therapeutic cloning. It is necessary to improve many techniques and reinforce the research of some basic theories, then somatic nuclear transfer and therapeutic cloning may apply to agriculture reproduction and benefit to human life better.

  13. Molecular Cloning and Expression of Human Interleukin-6 in Insect Cells

    Institute of Scientific and Technical Information of China (English)

    赵春文; 王嘉玺; 肖定华; 马贤凯

    1994-01-01

    670-bp hIL-6 cDNA fragments have been amplified by polymerase chain reaction(PCR)using recombinant plasmid pBMIL-6A as templates and two synthetic oligonucleotides containing the opti-mired translation initiation sequence/and restriction sites suitable for cloning as primers.The amplified IL-6cDNA fragments have then been recombined with a non-fusion expression baculovirus vector pVL1393.Theresultant recombinant plasmid pVL.IL-6 together with wtAcMNPV DNAs were transferred into culturedlepidopteran insect cells(Sf9)by calcium phosphate coprecipitation procedure.The recombinant baculovirus-es were formed by homologous recombination in vivo between pVL.IL-6 and wtAcMNPV DNAs,screenedfor plaque assay,and identified by means of dot blotting hybridization.The expressed rhIL-6 was secretedinto the culture medium,and its bioactivity was measured through half-maximum H-TdR incorporation intoIL-6-dependent cells 7TD1.As a result,the supernatant collected from recombinant baculovirus rAc.IL-6-infected Sf9 cells showed IL-6 activity of 10~6U/mL.The expression level of rhIL-6 of the supernatant deter-mined by IL-6 ELISA quantitation kit was 1 μg/mL.

  14. Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Hai-Yan Hu; Xiao-Ming Wang; Wei Wang; Xian-Xi Liu; Chun-Ying Jiang; Yan Zhang; Ji-Feng Bian; Yi Lu; Zhao Geng; Shi-Lian Liu; Chuan-Hua Liu

    2003-01-01

    AIM: To construct and express ODC recombinant gene for further exploring its potential use in early diagnosis of colorectal carcinoma.METHODS: Total RNA was extracted from colon cancer tissues and amplified by reverse-transcription PCR with two primers, which span the whole coding region of ODC. The synthesized ODC cDNA was cloned into vector pQE-30 at restriction sites BamH I and Sal I which constituted recombinant expression plasmid pQE30-ODC. The sequence of inserted fragment was confirmed by DNA sequencing,the fusion protein including 6His-tag was facilitated for purification by Ni-NTA chromatographic column.RESULTS: ODC expression vector was constructed and confirmed with restriction enzyme digestion and subsequent DNA sequencing. The DNA sequence matching on NCBI Blast showed 99 % affinity. The vector was transformed into E.coli M15 and expressed. The expressed ODC protein was verified with Western blotting.CONCLUSION: The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.

  15. PERSPECTIVAS SANITARIAS PROMETEDORAS DEL CLONAJE HUMANO PERSPECTIVAS SANITARIAS PROMETEDORAS DA CLONAGEM HUMANO PROMISING HEALTH PERSPECTIVES FOR HUMAN CLONING

    Directory of Open Access Journals (Sweden)

    Fermín Rolando Schramm

    2003-01-01

    clonagem através da análise racional e imparcial das implicações morais de seu uso e, se este for considerado legítimo, propôlo como meio para proteger a saúde humana. Apesar de ter aparentemente funções muito diferentes, tanto a clonagem terapêutica como a reprodutiva buscam responder ao desafio do sofrimento humano não necessário: a clonagem terapêutica, graças à técnica que utiliza célulastronco totipotentes, busca produzir órgãos e tecidos para eliminar o sofrimento evitável; a clonagem reprodutiva pode também eliminar formas de sofrimento humano evitáveis e pode, portanto, ser considerada um caso particular da clonagem terapêuticaTo evaluate the promises of human cloning, both therapeutic and reproductive, we can start from two pertinent points of view: health and secular bioethics. Under the point of view of health, though public prevention and health promotion policies, adequate to objective existing conditions, they try to protect human population from unnecessary diseases. Secular bioethics seeks to understand the ethics of cloning through rational analysis, free from the moral implications of its use; and if the conclusion is that it is legitimate, to propose it like a tool to protect human health. In spite of their apparently very different functions, both therapeutic cloning and reproductive cloning seek to respond to the challenge of unnecessary human suffering: therapeutic cloning, thanks to the technique that uses totipotent stem cells, seek to grow organs and tissues to eliminate avoidable suffering; reproductive cloning may also diminish forms of unnecessary human suffering and if can, therefore, to be considered a particular case of therapeutic approach

  16. Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression.

    Science.gov (United States)

    Kim, Geon A; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Oh, Hyun Ju; Hwang, Joing-Ik; Ahn, Curie; Saadeldin, Islam M; Lee, Byeong Chun

    2017-01-01

    Soluble human tumor necrosis factor (shTNFRI-Fc) and human heme oxygenase 1 (hHO-1) are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (P hHO-1 piglets (P hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

  17. Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression

    Directory of Open Access Journals (Sweden)

    Geon A. Kim

    2017-01-01

    Full Text Available Soluble human tumor necrosis factor (shTNFRI-Fc and human heme oxygenase 1 (hHO-1 are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. Also, H2O2 contents were increased, and superoxide dismutase was significantly lower in the higher copy number of shTNFRI-Fc and hHO-1 piglets (P<0.05. These results indicate that TNFRI-Fc and hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism.

  18. Cloning, Expression and Functional Characterization of In-House Prepared Human Basic Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    Hassan Rassouli

    2013-01-01

    Full Text Available Objective: Human basic fibroblast growth factor (bFGF plays an important role in cellular proliferation, embryonic development, and angiogenesis as well as in several signaling pathways of various cell types. bFGF is an essential growth factor for the maintenance of undifferentiated human embryonic stem cells (hESCs and human induced pluripotent stem cells (hiPSCs.Materials and Methods: In this experimental study, we present a straightforward method to produce biologically active recombinant human bFGF protein in E. coli that has long-term storage ability.Results: This procedure provides a rapid, cost effective purification of a soluble human bFGF protein that is biologically active and functional as measured in hESCs and hiPSCs in vitro and in vivo.Conclusion: The results show no significant difference in function between our in-house produced and commercialized bFGF.

  19. Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans

    Directory of Open Access Journals (Sweden)

    Stahl Stefanie

    2002-02-01

    Full Text Available Abstract Background Mucolipidosis type IV (MLIV is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. Results The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. Conclusions Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

  20. Dissemination of antimicrobial-resistant clones of Salmonella enterica among domestic animals, wild animals, and humans.

    Science.gov (United States)

    Palomo, Gonzalo; Campos, Maria Jorge; Ugarte, María; Porrero, María Concepción; Alonso, Juan Manuel; Borge, Carmen; Vadillo, Santiago; Domínguez, Lucas; Quesada, Alberto; Píriz, Segundo

    2013-02-01

    Non-typhoidal salmonellosis is an important zoonotic disease caused by Salmonella enterica. This work focuses on the identification of Salmonella enterica clonal strains which, presenting a wide distribution potential, express resistance determinants that compromise effectiveness of the antimicrobial therapy. The screening was performed on 506 Salmonella enterica isolates from animals and humans, which were characterized by serovar and phage typing, genome macrorestriction and pulsed-field gel electrophoresis, and detection of phenotypic and genotypic traits for antimicrobial resistance. A Salmonella Enteritidis strain with strong quinolone resistance is spread on three host environments carrying one of the four variants found for the GyrA protein: (1) Asp87Tyr, the major polymorphism found in 39 Salmonella isolates from human origin and six from poultry; (2) Ser83Phe, with four isolates from human origin and one from white stork (Ciconia ciconia); and (3) Asp87Asn or (4) Asp87Gly, with two isolates each from human origins. Several Salmonella Typhimurium strains that presented int1 elements and the classically associated pentaresistance (ACSSuT) phenotype were found distributed between two host environments: domestic animals and humans, domestics and wild animals, or wild fauna plus humans. This study points out the importance of monitoring gut microbiota and its antimicrobial resistance from wildlife, in parallel to livestock animals and humans, especially for animal species that are in close contact with people.

  1. Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity

    DEFF Research Database (Denmark)

    Vorum, H; Liu, X; Madsen, Peder;

    1998-01-01

    By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No. IEF SSP 9302 as the human homologue of calumenin. The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin. The deduced protein contains a 19 amino acid ...

  2. Comparative analysis of a BAC contig of the porcine RN region and the human transcript map: implications for the cloning of trait loci.

    Science.gov (United States)

    Jeon, J T; Amarger, V; Rogel-Gaillard, C; Robic, A; Bongcam-Rudloff, E; Paul, S; Looft, C; Milan, D; Chardon, P; Andersson, L

    2001-03-15

    The poorly developed transcript maps and the limited resources for genome analysis hamper positional cloning of trait loci in farm animals. This study demonstrates that this will now be easier by the combined use of BAC contigs and the import of the near complete human transcript map. The conclusion was obtained by a comparative analysis of a 2.4-Mb BAC contig of the RN region in pigs. The contig was constructed as part of a successful positional cloning project, which identified PRKAG3 as the causative gene for the RN phenotype. A comparative map including the corresponding regions on human chromosome 2q35 and mouse chromosome 1 (region 36-44 cM) is reported. Sixteen coding sequences were mapped on the BAC contig. The majority of these were identified by BLAST searches of BAC end sequences and BAC shotgun sequences generated during the positional cloning project. Map data for the orthologues in humans were available for 12 of the 16 coding sequences, and all 12 have been assigned to 2q35. Furthermore, no evidence for any rearrangement in gene order was obtained. The extensive linkage conservation indicates that the near complete human transcript map will be an invaluable resource for positional cloning projects in pigs and other domestic animals.

  3. The Budapest Meeting 2005. Intensified networking on ethics of science : The case of reproductive cloning, germline gene therapy and human dignity

    NARCIS (Netherlands)

    van Steendam, Guido; Dinnyes, Andras; Mallet, Jacques; Roosendaal, Hans E.

    2006-01-01

    This paper reports on the meeting of the Sounding Board of the EU Reprogenetics Project that was held in Budapest, Hungary, 6–9 November 2005. The Reprogenetics Project runs from 2004 until 2007 and has a brief to study the ethical aspects of human reproductive cloning and germline gene therapy.

  4. The Budapest Meeting 2005. Intensified networking on ethics of science : The case of reproductive cloning, germline gene therapy and human dignity

    NARCIS (Netherlands)

    Steendam, van Guido; Dinnyes, Andras; Mallet, Jacques; Roosendaal, Hans E.

    2006-01-01

    This paper reports on the meeting of the Sounding Board of the EU Reprogenetics Project that was held in Budapest, Hungary, 6–9 November 2005. The Reprogenetics Project runs from 2004 until 2007 and has a brief to study the ethical aspects of human reproductive cloning and germline gene therapy. Dis

  5. The Budapest Meeting 2005. Intensified networking on ethics of science : The case of reproductive cloning, germline gene therapy and human dignity

    NARCIS (Netherlands)

    van Steendam, Guido; Dinnyes, Andras; Mallet, Jacques; Roosendaal, Hans E.

    2006-01-01

    This paper reports on the meeting of the Sounding Board of the EU Reprogenetics Project that was held in Budapest, Hungary, 6–9 November 2005. The Reprogenetics Project runs from 2004 until 2007 and has a brief to study the ethical aspects of human reproductive cloning and germline gene therapy. Dis

  6. Transfection of the cloned human excision repair gene ERCC-1 to UV-sensitive CHO mutants only corrects the repair defect in complementation group 2 mutants.

    NARCIS (Netherlands)

    M. van Duin (Mark); J.H. Janssen; J. de Wit (Jan); J.H.J. Hoeijmakers (Jan); L.H. Thompson; D. Bootsma (Dirk); A. Westerveld (Andries)

    1988-01-01

    textabstractThe human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In ord

  7. Arguments for and against human cloning in terms of teleological and deontological theories

    National Research Council Canada - National Science Library

    Ionut Stefan

    2015-01-01

    .... Mill, while the Kantian perspective is exemplary for deontological ethics. According to the teleological perspective, a form of human behavior is described as moral or non-moral according to the goals explicitly set...

  8. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  9. Cloning and characterization of human IC53-2, a novel CDK5 activator binding protein

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    We have identified IC53-2, a human homologue of the rat C53 gene from a human placenta cDNA library (GeneBank Accession No. AF217982). IC53-2 can bind to the CDK5 activator p35 by in vitro association assay. IC53-2 is mapped to human chromosome 17q21.31. The IC53-2 transcript is highly expressed in kidney, liver, skeletal muscle and placenta. It is abundantly expressed in SMMC-7721, C-33A, 3AO, A431and MCF-7 cancer cell lines by RT-PCR assay. Stable transfection of IC53-2 cDNA into the hepatocellularcarcinoma SMMC-7721 cell remarkably stimulates its growth in vitro. The above results indicate thatIC53-2 is a novel human gene, which may be involved in the regulation of cell proliferation.

  10. Molecular cloning and characterization of a novel human kinase gene, PDIK1L

    Indian Academy of Sciences (India)

    Lingchen Guo; Chaoneng Ji; Shaohua Gu; Kang Ying; Haipeng Cheng; Xiaoghua Ni; Jianping Liu; Yi Xie; Yumin Mao

    2003-04-01

    We isolated a 4301-bp cDNA from a human foetal brain cDNA library by high-throughput cDNA sequencing. It encodes a protein of 341 amino acids, which shows 69% identity with the human kinase CLIK1 (AAL99353), which was suggested to be the CLP-36 interacting kinase. Bioinformatics analysis suggests that the putative kinase may interact with PDZ and LIM domain proteins. Therefore the protein and its cDNA were named ‘PDLIM1 interacting kinase 1 like’ (PDIK1L; nomenclature approved by the HUGO Gene Nomenclature Committee). Ensembl Genome Browser located PDIK1L to human chromosome 1p35.3. It spans about 13.7 kb and consists of four exons and three introns. Multiple-tissue cDNA panel PCR revealed that the gene is expressed widely in human tissues: liver, kidney, pancreas, spleen, thymus and prostate. The protein appears to be localized to the nucleus.

  11. Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

    Science.gov (United States)

    Sano, Akiko; Matsushita, Hiroaki; Wu, Hua; Jiao, Jin-An; Kasinathan, Poothappillai; Sullivan, Eddie J; Wang, Zhongde; Kuroiwa, Yoshimi

    2013-01-01

    Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

  12. Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle.

    Directory of Open Access Journals (Sweden)

    Akiko Sano

    Full Text Available Therapeutic human polyclonal antibodies (hpAbs derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc cattle carrying a human artificial chromosome (HAC comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH and kappa-chain (hIGK germline loci (named as κHAC are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO. However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ in the pre-B cell receptor (pre-BCR complex, we partially replaced (bovinized the hIgM constant domain with the counterpart of bovine IgM (bIgM that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO, the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG can be produced from such Tc cattle.

  13. Human Vascular Endothelial Growth Factor cDNA Cloning and Expression in Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Human vascular endothelial growth factor (VEGF) cDNA was amplified by nested polymerase chain reaction method from the HL60 cells. Then a pCD-hVEGF165 recombinant plasmid was constructed. Rabbit osteoblasts were transfected with pCD-hVEGF165 plasmid by lipofectin mediated gene transfer. The transient expressive results were detected by immunohistochemical method. It was observed that the expression of human VEGF gene was detected 72 h after transfecting distinctly.

  14. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Directory of Open Access Journals (Sweden)

    Dipak K. Dube

    2016-01-01

    Full Text Available In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4 each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

  15. Cloning and characterization of a human delayed rectifier potassium channel gene.

    Science.gov (United States)

    Albrecht, B; Lorra, C; Stocker, M; Pongs, O

    1993-01-01

    A human genomic DNA library was screened for sequences homologues to the rat delayed rectifier Kv 2.1 (DRK1) K+ channel cDNA. Three phages were isolated which hybridized to Kv 2.1 cDNA probes. Alignment of the human genomic DNA sequence with the rat cDNA sequence indicated that the open reading frame (ORF) is interrupted by a large intervening sequence, that separates exons encoding the membrane spanning core region of the K+ channel polypeptide. The Kv 2.1 gene occurs once in the human genome and has been mapped to chromosome 20. The human, mouse and rat Kv 2.1 proteins have been highly conserved, showing only a few substitutions outside of the membrane spanning domains in the amino- and carboxy-terminal cytoplasmic domains. Nevertheless, expression of human DRK1 channels in Xenopus oocytes showed that mouse, rat and human Kv 2.1 channels have distinct pharmacological and electrophysiological properties. The observed differences in activation, voltage-dependence, 4-aminopyridine sensitivity and single-channel conductance have to be attributed to amino acid substitutions in the amino-and/or carboxy-terminal cytoplasmic domains. Obviously, these domains of Kv 2.1 channels influence biophysical K+ channel properties, which are thought to be determined solely by the membrane spanning core domain of potassium channels.

  16. Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans

    Science.gov (United States)

    Abbott, Lynn; Alshiekh-Nasany, Ruham; Mitschow, Charles

    2016-01-01

    In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart. PMID:27703814

  17. To clone or not to clone--whither the law?

    Science.gov (United States)

    Lupton, M L

    1999-01-01

    The cloning of Dolly the lamb from adult cells by scientists at the Roslin Laboratories near Edinburgh in February 1997 has startled the world because it now opens the way to clone adult human beings. The reaction to Ian Wilmut's breakthrough has been instant and largely negative. Bills were rushed into both the US Senate and House of Representatives aimed at banning the cloning of human beings. Human cloning is premature at this stage, but there are many positive spin-offs of cloning in the field of genetic engineering, such as the production of human proteins such as blood clotting factors which aid in healing wounds. Progress by means of cloning can also be made into devising a cure for Parkinson's Disease amongst others. No lesser ethicist than John C. Fletcher of the University of Virginia foresees circumstances in which human cloning is acceptable e.g. to enable a couple to replace a dying child, to enable a couple, one of whom is infertile, to clone a child from either partner. Extensive regulation of cloning by the law is inevitable but, in doing so, the legislation should be careful not to outlaw research in this area which could be beneficial to mankind.

  18. Chromosomal mapping of 18S-28S rRNA genes and 10 cDNA clones of human chromosome 1 in the musk shrew (Suncus murinus).

    Science.gov (United States)

    Kuroiwa, A; Matsubara, K; Nagase, T; Nomura, N; Seong, J K; Ishikawa, A; Anunciado, R V; Tanaka, K; Yamagata, T; Masangkay, J S; Dang, V B; Namikawa, T; Matsuda, Y

    2001-01-01

    The direct R-banding fluorescence in situ hybridization (FISH) method was used to map 18S-28S ribosomal RNA genes and 10 human cDNA clones on the chromosomes of the musk shrew (Suncus murinus). The chromosomal locations of 18S-28S ribosomal RNA genes were examined in the five laboratory lines and wild animals captured in the Philippines and Vietnam, and the genes were found on chromosomes 5, 6, 9, and 13 with geographic variation. The comparative mapping of 10 cDNA clones of human chromosome 1 demonstrated that human chromosome 1 consisted of at least three segments homologous to Suncus chromosomes (chromosomes 7, 10, and 14). This approach with the direct R-banding FISH method is useful for constructing comparative maps between human and insectivore species and for explicating the process of chromosomal rearrangements during the evolution of mammals.

  19. Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.

    Science.gov (United States)

    Fridy, Peter C; Otto, James C; Dollins, D Eric; York, John D

    2007-10-19

    Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events.

  20. Molecular cloning of a human gene that is a member of the nerve growth factor family

    Energy Technology Data Exchange (ETDEWEB)

    Jones, K.R.; Reichardt, L.F. (Howard Hughes Medical Institute, San Francisco, CA (USA))

    1990-10-01

    Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors, nerve growth factor and brain-derived neurotrophic factor, share substantial amino acid sequence identity. The authors have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene, neurotrophin-3, that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain, suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development.

  1. Cloning and characterization of a novel member of human β-1,4-galactosyltransferase gene family

    Institute of Scientific and Technical Information of China (English)

    范玉新; 余龙; 张琪; 江萤; 戴方彦; 陈驰原; 屠强; 毕安定; 许月芳; 赵寿元

    1999-01-01

    By using the EST strategy for identifying novel members belonging to homologous gene families, a novel full-length cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GAlT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1032 nucleotides (344 amino acids). In view of the homology to members of the galactosyltransferase gene family and especially the closest relationship to Gallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1, 4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3.

  2. Gene cloning of human soluble CD14 and its expression in eucaryotic cells

    Institute of Scientific and Technical Information of China (English)

    荫俊; 白洁; 王威; 宋伟; 王忠泽

    2002-01-01

    To express human soluble CD14 (sCD14) in eukaryotic cells. Methods: Human sCD14 cDNA was amplified from U937 cells with RT-PCR method. The recombinant expression plasmid pEF1/HisC/sCD14348aa was constructed and the expression in COS-7 cells was carried out using liposome transfection method. The yield was examined with scanning map identification. The expressed product was purified by immuno-affinity chromatography. Results: Sequence analysis demonstrated that the amplified gene sequence and those reported by documents were completely identical. sCD14 was expressed with high-yield. The expressed product was purified to above 90%. Recombinant sCD14, specifically combinable with endotoxins, had a natural biological activity. Conclusions: Human sCD14 was expressed in COS-7 cells, which laid a foundation for further study.

  3. Molecular cloning and characterization of six novel isoforms of human Bim, a member of the proapoptotic Bcl-2 family.

    Science.gov (United States)

    U, M; Miyashita, T; Shikama, Y; Tadokoro, K; Yamada, M

    2001-11-30

    Bim protein is one of the BH3-only proteins, members of the Bcl-2 family that have only one of the Bcl-2 homology regions, BH3. BH3-only proteins are essential initiators of apoptotic cell death. Thus far, three isoforms of Bim have been reported, i.e. Bim(EL), Bim(L) and Bim(S). Here we report the cloning and characterization of six novel isoforms of human Bim, designated as Bimalpha1, alpha2, and beta1-beta4, which are generated by alternative splicing. Unlike the three known isoforms, none of these novel isoforms contained a C-terminal hydrophobic region. Among the novel isoforms, only Bimalpha1 and alpha2 contained a BH3 domain and were proapoptotic, although less potent than the classical isoforms. These two isoforms localized, at least in part, in mitochondria when transiently expressed in HeLa cells as a green fluorescent protein-fused form. These results suggest that the BH3 domain is necessary for induction of apoptosis and mitochondrial localization but not sufficient for the full proapoptotic activity. While the classical isoforms were always predominantly expressed in transformed cells, expression profiles of bim isoforms were highly variable among normal tissues at least in humans, suggesting a tissue-specific transcriptional regulation of bim.

  4. Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

    Institute of Scientific and Technical Information of China (English)

    An-LiJIANG; Jian-YeZHANG; CharlesYOUNG; Xiao-YanHU; Yong-MeiWANG; Zhi-FangLIU; Mei-LanHAO

    2004-01-01

    Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development and prostate cancer. To study its regulation of transcription, 1.06 kb 5′ flanking region of Nkx3.1 gene and its 5′ deletion mutants (861,617,417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, a promoter-less luciferase reporter vector, to examine their promoter activities driving the reporter gene transcription, pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL3-control and pGL3-basic were used as positive and negative control respectively. The promoter activities were determined by dual-luciferase reporter assay 48h after pGL3 constructs were cotransfected with pRL-TK into prostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M/M2) of pGL3-1.06kb cotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL3-control cotransfection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The results also showed that the relative activities (M1/M2) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861bp, pGL3-617bp, pGL3-417bp, pGL3-238bp, the last one still had 80% promoter activity compared with pGL3-1.06kb, which showed that deletion from 1.06kb to 238 bp had small effects on promoter activity. The conclusion was that the 238bp fragment containing a TATA box and two CAAT boxes had strong promoter activity. However, the deletion from 1.06kb to 861bp reduced activity 3.8-fold while the deletion from 417bp to 238bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain some important positive or negative regulatory elements. It will be important to identify the elements within the Nkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

  5. Molecular Cloning and Characterization of Human Homeobox Gene Nkx3.1 Promoter

    Institute of Scientific and Technical Information of China (English)

    An-Li JIANG; Jian-Ye ZHANG; Charles YOUNG; Xiao-Yan HU; Yong-Mei WANG; Zhi-Fang LIU; Mei-Lan HAO

    2004-01-01

    Nkx3.1 is a prostate-specific homeobox gene related strongly to prostate development andprostate cancer. To study its regulation of transcription, 1.06 kb 5 ′ flanking region of Nkx3.1 gene and its5 ′deletion mutants (861,617, 417 and 238 bp) were obtained by PCR and cloned into pGL3-basic, apromoter-less luciferase reporter vector, to examine their promoter activities driving the reporter genetranscription, pRL-TK, a Renilla luciferase reporter vector was used as internal control, and pGL3-controland pGL3-basic were used as positive and negative control respectively. The promoter activities were deter-mined by dual-luciferase reporter assay 48 h after pGL3 constructs were cotransfected with pRL-TK intoprostate cancer cell LNCaP. The results showed that dual-luciferase reporter assay (M1/M2) of pGL3-1.06 kbcotransfection with pRL-TK was 2.7, which was about 1.5-fold higher than that of pGL3-control cotrans-fection with pRL-TK and 50-fold higher than that of pGL3-basic cotransfection with pRL-TK. The resultsalso showed that the relative activities (M1/M2) were 0.71, 0.84, 0.44 and 2.07 respectively for pGL3-861 bp,pGL3-617 bp, pGL3-417 bp, pGL3-238 bp, the last one still had 80% promoter activity compared with pGL3-1.06 kb, which showed that deletion from 1.06 kb to 238 bp had small effects on promoter activity. Theconclusion was that the 238 bp fragment containing a TATA box and two CAAT boxes had strong promoteractivity. However, the deletion from 1.06 kb to 861 bp reduced activity 3.8-fold while the deletion from 417bp to 238 bp enhanced activity 4.7-fold, which indicated that these deleted sequences might contain someimportant positive or negative regulatory elements. It will be important to identify the elements within theNkx3.1 promoter that contribute to regulation of the gene transcription in the future studies.

  6. [Developing a physical map of human chromosome 22 using Pace electrophoresis and large fragment cloning]. Annual report, October 1, 1989--September 30, 1990

    Energy Technology Data Exchange (ETDEWEB)

    Simon, M.I.

    1990-12-31

    In the past year the authors have made significant progress in the development of a bacterial based cloning system for large fragments of mammalian DNA. They have completed construction of several recombination deficient bacterial host strains designed to minimize homologous recombination arising with repeats within cloned DNA. Despite the multiple mutations, these strains are viable and grow readily on standard media (LB). One of the chief attractions of a bacterial system is the promise of high transformation efficiencies. The author have pursued two separate strategies with the vector. The first makes use of the cos sites in the vector to package cloned DNA as phage particles for infection. By maintaining the vector as a single copy in the recombination minus host, they believe that the recombination that affects conventional cosmid libraries will be eliminated. They encountered no difficulties in preparing such a ``Fosmid`` (F factor based cosmid) library of human DNA.

  7. Artificial synthesis and cloning of human obese gene%人肥胖(obese)基因的人工合成及克隆

    Institute of Scientific and Technical Information of China (English)

    杨林; 吴无畏

    2000-01-01

    根据人肥胖基因的cDNA序列,通过合理的引物设计、链延伸反应、PCR反应以及分子克隆等步骤,成功地合成出编码瘦蛋白(Leptin)的肥胖基因(ob基因)全长片段,并将其克隆至pUC18载体质粒上.序列分析和酶切鉴定显示肥胖基因得到了正确合成和克隆.%According to the known sequence of human ob gene cDNA,the gene coding the leptin was synthesized and cloned into pUC18 vector after reasonable primer designing,DNA strain extending,PCR reaction and molecular cloning.Sequence analysis and RE digest results showed the ob gene was synthesized and cloned correctly.

  8. [Analysis, identification and correction of some errors of model refseqs appeared in NCBI Human Gene Database by in silico cloning and experimental verification of novel human genes].

    Science.gov (United States)

    Zhang, De-Li; Ji, Liang; Li, Yan-Da

    2004-05-01

    We found that human genome coding regions annotated by computers have different kinds of many errors in public domain through homologous BLAST of our cloned genes in non-redundant (nr) database, including insertions, deletions or mutations of one base pair or a segment in sequences at the cDNA level, or different permutation and combination of these errors. Basically, we use the three means for validating and identifying some errors of the model genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS: (I) Evaluating the support degree of human EST clustering and draft human genome BLAST. (2) Preparation of chromosomal mapping of our verified genes and analysis of genomic organization of the genes. All of the exon/intron boundaries should be consistent with the GT/AG rule, and consensuses surrounding the splice boundaries should be found as well. (3) Experimental verification by RT-PCR of the in silico cloning genes and further by cDNA sequencing. And then we use the three means as reference: (1) Web searching or in silico cloning of the genes of different species, especially mouse and rat homologous genes, and thus judging the gene existence by ontology. (2) By using the released genes in public domain as standard, which should be highly homologous to our verified genes, especially the released human genes appeared in NCBI GENOME ANNOTATION PROJECT REFSEQS, we try to clone each a highly homologous complete gene similar to the released genes in public domain according to the strategy we developed in this paper. If we can not get it, our verified gene may be correct and the released gene in public domain may be wrong. (3) To find more evidence, we verified our cloned genes by RT-PCR or hybrid technique. Here we list some errors we found from NCBI GENOME ANNOTATION PROJECT REFSEQs: (1) Insert a base in the ORF by mistake which causes the frame shift of the coding amino acid. In detail, abase in the ORF of a gene is a redundant insertion, which causes a reading frame

  9. Academic Cloning.

    Science.gov (United States)

    Sikula, John P.; Sikula, Andrew F.

    1980-01-01

    The authors define "cloning" as an integral feature of all educational systems, citing teaching practices which reward students for closely reproducing the teacher's thoughts and/or behaviors and administrative systems which tend to promote like-minded subordinates. They insist, however, that "academic cloning" is not a totally…

  10. Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

    Institute of Scientific and Technical Information of China (English)

    马凤蓉; 朱立平; 汪燚; 赵方萄; 史耕先; 李波; 李国燕; 张淑珍; 王讯

    2000-01-01

    A 1 846 bp cDNA is isolated from a human tonsil cell λgt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.

  11. "Asexual propagation of a virulent clone complex in human and feline outbreak of sporotrichosis"

    NARCIS (Netherlands)

    Teixeira, Marcus de Melo; Rodrigues, Anderson Messias; Tsui, Clement K M; de Almeida, Luiz Gonzaga Paulo; Van Diepeningen, Anne D; Gerrits van den Ende, Bert; Fernandes, Geisa Ferreira; Kano, Rui; Hamelin, Richard C; Lopes-Bezerra, Leila Maria; Ribeiro Vasconcelos, Ana Tereza; de Hoog, Sybren; de Camargo, Zoilo Pires; Felipe, Maria Sueli Soares

    2014-01-01

    Sporotrichosis is one of the most frequent subcutaneous fungal infections to human and animals caused by members of the plant-associated, dimorphic genus Sporothrix. Three of the four medically important Sporothrix species found in Brazil have been considered asexual, as no sexual stage has ever bee

  12. Molecular cloning and pharmacology of functionally distinct isoforms of the human histamine H(3) receptor

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Goodman, M W; Burstein, E S

    2002-01-01

    The pharmacology of histamine H(3) receptors suggests the presence of distinct receptor isoforms or subtypes. We herein describe multiple, functionally distinct, alternatively spliced isoforms of the human H(3) receptor. Combinatorial splicing at three different sites creates at least six distinc...

  13. Cloning and expression of a novel human profilin variant, profilin II

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Andersen, A H

    1993-01-01

    We have isolated a 1.7 kbp cDNA encoding a 140 amino acid protein (15.1 kDa, pI 5.91) with a high sequence similarity (62%) to human profilin (profilin I). We have termed this variant profilin II. Northern blot analysis showed that profilin II is highly expressed in brain, skeletal muscle and kid...

  14. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship

  15. Genomic structure and cloning of two transcript isoforms of human Sp8.

    NARCIS (Netherlands)

    M.A. Milona (Maria-athina); J.E. Gough (Julie); A.J. Edgar (Alasdair)

    2004-01-01

    textabstractBACKGROUND: The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been characte

  16. Cloning and characterization of the human DNA-excision repair gene ERCC-1

    NARCIS (Netherlands)

    M. van Duin (Michel)

    1988-01-01

    textabstractIt is the aim of the work described in this thesis to isolate and characterize human genes involved DNA excision repair. This will facilitate the understanding of the mechanism of this repair process whereas it also provides an important step to better understand the relationship between

  17. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and variab

  18. Cloning, expression, and chromosomal assignment of the human mitochondrial intermediate peptidase gene (MIPEP).

    Science.gov (United States)

    Chew, A; Buck, E A; Peretz, S; Sirugo, G; Rinaldo, P; Isaya, G

    1997-03-15

    The mitochondrial intermediate peptidase of Saccharomyces cerevisiae (YMIP) is a component of the yeast mitochondrial protein import machinery critically involved in the biogenesis of the oxidative phosphorylation (OXPHOS) system. This leader peptidase removes specific octapeptides from the amino terminus of nuclear-encoded OXPHOS subunits and components of the mitochondrial genetic apparatus. To address the biologic role of the human peptidase [MIPEP gene, HMIP polypeptide], we have initiated its molecular and functional characterization. A full-length cDNA was isolated by screening a human liver library using a rat MIP (RMIP) cDNA as a probe. The encoded protein contained a typical mitochondrial leader peptide and showed 92 and 54% homology to RMIP and YMIP, respectively. A survey of human mitochondrial protein precursors revealed that, similar to YMIP, HMIP is primarily involved in the maturation of OXPHOS-related proteins. Northern analysis showed that the MIPEP gene is differentially expressed in human tissues, with the highest levels of expression in the heart, skeletal muscle, and pancreas, three organ systems that are frequently affected in OXPHOS disorders. Using fluorescence in situ hybridization, the MIPEP locus was assigned to 13q12. This information offers the possibility of testing the potential involvement of HMIP in the pathophysiology of nuclear-driven OXPHOS disorders.

  19. [The drinking problem--temperance after prohibition].

    Science.gov (United States)

    Hamran, Olav

    2005-12-15

    The alcohol issue had a prominent place in Norwegian politics and society in the first decades of the 20th century; teetotalers' organisations had a major role. Prohibition was the ultimate way of solving the alcohol problem. After the prohibition (1916-27), the teetotalers lost their hegemony. Anti-prohibition views became influential in the making of Norwegian alcohol policy. The post-prohibition years have been seen as at period of change in which a moralist and condemning attitude to alcohol and alcoholics were replaced by a more humane disease concept of alcoholism. This article challenges this view. After prohibition, to separate between use and abuse of alcohol was the main focus of Norwegian alcohol policy. Alcohol abuse and alcohol abusers became the main targets. Abuse of alcohol was first and foremost seen as a social category, as immoral behaviour. In the 1930s, a moralist and condemning attitude toward excessive drinking and drinkers was put forward, not replaced, in Norwegian alcohol policy. A concept of alcoholism as a disease had little or no impact in these years.

  20. Cloning, expression and mapping of the full-length cDNA of human CCTβ subunit

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Chaperonins assist the proper folding of target proteins without being a part of the substrates. The eukaryotic cytosolic chaperonin, CCT-Chaperonin Containing TCP-1 (tailless complex polypeptide-1), is mainly involved in the formation of cytoskeletal proteins and is essential for cell viability. Mammalian CCT is commonly a protein complex composed of 7-9 subunit species. We have isolated a novel full-length cDNA from human testis cDNA library. This cDNA of 1935 bp contains a 1605 bp open reading frame (ORF) encoding 535 amino acids (aa). The deduced protein of the cDNA is highly homologous to the CCTβ subunit of saccharomyces cerevisiae, schizosaccharomyces pombe, caenorhabditis elegans and mouse, etc. Especially high homology (97%) is found between the deduced protein and mouse CCTb. On the basis of such high homology, the protein encoded by the new gene was proposed to be a human CCTβ subunit. Northern hybridization showed that human CCTβ gene is expressed as a transcript of about 2.0 kb in various tissues. Overexpression was seen in testis with the expression level 3-24 times of those in other tissues. The CCTβ gene was mapped to human chromosome 12q14 by Radiation Hybrid Mapping. Through homologous search, the 5′-end of the cDNA sequence was found to share intermittent regional homology with the 3′-end of human genomic sequence (U91327). The genomic structure of the 5′-end of CCTβ was also described in detail through comparative analysis.

  1. Ethical issues in livestock cloning.

    Science.gov (United States)

    Thompson, P B

    1999-01-01

    Although cloning may eventually become an important technology for livestock production, four ethical issues must be addressed before the practice becomes widespread. First, researchers must establish that the procedure is not detrimental to the health or well-being of affected animals. Second, animal research institutions should evaluate the net social benefits to livestock producers by weighing the benefits to producers against the opportunity cost of research capacity lost to biomedical projects. Third, scientists should consider the indirect effects of cloning research on the larger ethical issues surrounding human cloning. Finally, the market structure for products of cloned animals should protect individual choice, and should recognize that many individuals find the prospect of cloning (or consuming cloned animals) repugnant. Analysis of these four issues is complicated by spurious arguments alleging that cloning will have a negative impact on environment and genetic diversity.

  2. Production of human lysozyme-transgenic cloned porcine embryos by somatic nuclear transfer

    Institute of Scientific and Technical Information of China (English)

    Qiuyan Li; Hengxi Wei; Ying Guo; Yan Li; Rui Zhao; Yufang Ma; Zhengquan Yu; Bo Tang; Lei Zhang; Yunping Dai; Ning Li

    2009-01-01

    Due to their physiology and organ size, pigs have significant potential as human disease models and as organ transplantation donors. Genetic modification of pigs could provide benefits for both agriculture and human medicine. In this study, five fetal pig fibroblast cell lines from two species (Wuzhishan and Landrace pigs) were transfected using double-marked human lysozyme (HLY) plasmids (pBC1-HLY-GFP-NEO) by a liposome-mediated method. The ratio of green fluorescent protein (GFP)-expressing cells was >95% in sw7, sw8, s1w3 and s1w6 cell lines, but only 49.3% in slw9 cells. Cells from the four highly transgenic lines were used as nuclear donors to construct embryos, which were then cultured after fusion and activation by electric stimulation. The rate of cleavage was 76.7%, 48 h after acti-vation. After 7 days, 18.5% of cleaved eggs had developed to the blastocyst stage and 93.3% of blastocysts were GFP-positive. These results indicate that transgenic fetal pig fibroblast cell lines could be obtained by a liposome-mediated method, though the transfection efficiency varied between cell lines. Reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive.

  3. Human augmenter of liver regeneration: molecular cloning, biological activity and roles in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    杨晓明; 谢玲; 邱兆华; 吴祖泽; 贺福初

    1997-01-01

    The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA . The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87% ) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an im-portant parac

  4. Cloning and identification of a novel human glioma differentiation related gene GDR1

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed gene GDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame of GDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows that GDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns of GDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.

  5. Cloning and expression of recombinant human platelet-derived growth factor-BB in Pichia Pink.

    Science.gov (United States)

    Babavalian, H; Latifi, A M; Shokrgozar, M A; Bonakdar, S; Tebyanian, H; Shakeri, F

    2016-07-31

    The PDGF-BB plays a key role in several pathogenesis diseases and it is believed to be an important mediator for wound healing. The recombinant human PDGF-BB is safe and effective to stimulate the healing of chronic, full thickness and lower extremity diabetic neurotrophic ulcers. In the present study, we attempted to produce a PDGF-BB growth factor and also, evaluate its functionality in cell proliferation in yeast host Pichia pink. Pichia pink yeast was used as a host for evaluation of the rhPDGF-BB expression. The coding sequence of PDGF-BB protein was synthesized after optimization and packed into the pGEM. Recombinant proteins were produced and purified. The construct of pPinkα-HC-pdgf was confirmed by sequence, the PDGF-BB protein was expressed and purified with using a nickel affinity chromatography column and then characterized by SDS-PAGE electrophoresis. The biological activity of PDGF-BB was estimated with using human fibroblast cell line. The measurement of protein concentration was determined by Bradford and human PDGF-BB ELISA kit. Purified rhPDGF-BB showed similar biological activity (as the standard PDGF-BB) and suggested that the recombinant protein has a successful protein expression (as well as considerable biological activity in P. pink host). The exact amount of recombinant PDGF-BB concentrations were measured by specific ELISA test which it was about 30 μg/ml. Our study suggested that efficiency of biological activity of PDGF-BB protein may be related to its conformational similarity with standard type and also, it practically may be important in wound healing and tissue regeneration.

  6. Molecular Cloning of the Human Genes(s) Directing the Synthesis of Nervous System Cholinesterases.

    Science.gov (United States)

    1985-12-01

    it . ad tmir k ! , U.Z. Littaer, Y. Dudai, I. Dilwn, V.I. Taidtiirg and Z. %tgsl, Edo. pp. 201-215. Johnr wiley and Scm, Now York. 40. Razon , N...681-695. 41. Gurwitz, D., Razon , N., Sdoolovsky, M. and Soreq, H. (1984) Ja ren of muscarinic receptor in primary brain tumors. Dev. Brain ese. 14...growth factor receptors in human brairki ttumors. Canc~er Res. 44, 753-760. 69. Soreq, H., Zevin-Sorin, D. and Razon , N. (1984) Eqpressicn of cho

  7. 45 CFR 1326.11 - Prohibition against supplantation.

    Science.gov (United States)

    2010-10-01

    ... SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES THE ADMINISTRATION ON AGING, OLDER AMERICANS PROGRAMS GRANTS TO INDIAN TRIBES FOR SUPPORT AND NUTRITION SERVICES § 1326.11 Prohibition against supplantation. A...

  8. Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene.

    Science.gov (United States)

    Hsu, Cary; Jones, Stephanie A; Cohen, Cyrille J; Zheng, Zhili; Kerstann, Keith; Zhou, Juhua; Robbins, Paul F; Peng, Peter D; Shen, Xinglei; Gomes, Theotonius J; Dunbar, Cynthia E; Munroe, David J; Stewart, Claudia; Cornetta, Kenneth; Wangsa, Danny; Ried, Thomas; Rosenberg, Steven A; Morgan, Richard A

    2007-06-15

    Malignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8(+) T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus-based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28(-), CD45RA(-), CD45RO(+), and CD62L(-), a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen-specific T-cell receptors, the clone secreted IFN-gamma upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15R alpha expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation.

  9. Transforming Growth Factor-β2 Gene Cloning and Protein Expression in Human Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    曹阳; 魏厚仁; 笪邦红; 李忠玉

    2003-01-01

    Whether cultured human trabecular meshwork cells express transforming growth factor-β2 (TGF-β2) messenger RNA (mRNA) and protein was investigated. Total RNA of 106 cultured human trabecular meshwork cells was extracted with TRIZOL reagent, reverse transcriptase-polymerase chain reaction (RT-PCR) were used for detection of TGF-β2 messenger RNA, and the PCRproduct was verified by sequencing. Immunohistochemical staining was used to detect TGF-β2 protein. The results showed that a single RT-PCR amplified product was obtained, and the sequence was homologous to the known sequence. TGF-β2 immunostaining was positive. It was concluded that trabecular meshwork cells could produce TGF-β2 and contribute to the presence of TGF-β2 in trabecular meshwork microenvironment as well as aqueous humor. Trabecular meshwork cells were affected by TGF-β2 not only through paracrine, but also autocrine action. Whether abnormal changes in TGF-β2 production contribute to the pathogenesis of primary open-angle glaucoma is worth further in vestigation.

  10. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. No obvious bands were detected in other examined tissues. The GGPPS gene was located on human chromosome 1q43 by Radiation Hybrid mapping method. It was proved that there was a putative predisposing gene for prostate cancer in this region, and that analogs of GGPP can inhibit the geranylgeranylation of p21rap protein in PC-3 prostate cancer cell lines. These facts suggest that GGPPS may be one of the candidate genes for prostate cancer.

  11. Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein b-5

    Institute of Scientific and Technical Information of China (English)

    XIA; Jiahui; (夏家辉); ZHENG; Duo; (郑多),; TANG; Dongsheng; (唐冬生); DAI; Heping; (戴和平); PAN; Qian; (潘乾); LONG; Zhigao; (龙志高); LIAO; Xiaodong; (廖晓东)

    2001-01-01

    By homologous EST searching and nested PCR a new human gene GJB5 encoding gap junction protein b-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.

  12. Cloning, mapping and mutation analysis of human gene GJB5 encoding gap junction protein b-5

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    By homologous EST searching and nested PCR a new human gene GJB5encoding gap junction protein b-5 was identified. GJB5 was genetically mapped to human chromosome 1p33-p35 by FISH. RT-PCR revealed that it was expressed in skin, placenta and fetal skin. DNA sequencing of GJB5 was carried out in 142 patients with sensorineural hearing impairment and probands of 36 families with genetic diseases, including erythrokeratodermia (5 families), Charcot-Marie-Tooth disease (13), ptosis (4), and retinitis pigmentosa and deafness (14). Two missense mutations (686A→G, H229R; 25C→T, L9F) were detected in two sensorineural hearing impairment families. A heterologous deletion of 18 bp within intron was found in 3 families with heredity hearing impairment, and in one of the 3 families, a missense mutation (R265P) was identified also. But the deletion and missense mutation seemed not segregating with hearing impairment in the family. No abnormal mRNA or mRNA expression was detected in deletion carriers by RT-PCR analysis in skin tissue. Mutation analysis in 199 unaffected individuals revealed that two of them were carriers with the same 18 bp deletion.

  13. cDNA cloning and expression of an apoptosis-related gene, human TFAR15 gene

    Institute of Scientific and Technical Information of China (English)

    王玉刚; 刘洪涛; 张颖妹; 马大龙

    1999-01-01

    By means of cDNA-RDA method. some cDNA fragments were found to have high levels of expression during deprivation of GM-CSF (granulocyte macrophage-colony stimulating factor) in a human myeloid cell line, TF-1 cells. One of these tragments was identified as a novel gene. To get the full length of cDNA, rapid amplification of cDNA ends (RACE) and expressed sequence tags (EST) overlapping fragments assembling strategies were used. The novel gene was named TRAF15 (TF-1 cell apoptosis related gene-15), which consists of 1218 nueleotides and encodes 212 amino acids. The putative protein protein product of TFAR15 is partially homologous to C. elegans protein C14A4. 11. TFAR15 mRNA is expressed in fetal liver, kidney, spleen and lung. and also in some human myeloid cell lines. Both of the TFAR15 mRNA and protein were highly expressed in TF-(?) cells after GM-CSF withdrawal. In vitro analysis showed that the recombinant TFAR15 protein co(?)ld inhibit the natural cell death of 293 cells, an embryonic kidney cell

  14. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase.

    Science.gov (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C

    1986-01-01

    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  15. Cloning, expression, and purification of a synthetic human growth hormone in Escherichia coli using response surface methodology.

    Science.gov (United States)

    Zamani, Mozhdeh; Berenjian, Aydin; Hemmati, Shiva; Nezafat, Navid; Ghoshoon, Mohammad Bagher; Dabbagh, Fatemeh; Mohkam, Milad; Ghasemi, Younes

    2015-03-01

    The aim of this study was to achieve high-level production of the human growth hormone (hGH) in the prokaryotic expression system. In this regard, we performed cloning, expression, and purification of a synthetic hGH gene in BL21 (DE3) strain of E. coli. The hGH production was determined by SDS-PAGE and western blotting techniques, and then the protein concentration was determined by the Bradford assay. To gain insight into the effect of different nutrients on the growth of E. coli and hGH production, in a preliminary assessment nine different types of the basal medium were analyzed. The highest growth of E. coli and hGH production were observed in TB and SOB media. Accordingly, design of experiments was employed for screening the most significant nutrients, and central composite face design was applied for the optimization. The optimum medium consisted of yeast extract (10 g/L), tryptone (10 g/L), and K2HPO4 (2 g/L). The optimum hGH concentration was 391 mg/L, which was 3-fold higher than the hGH concentration in the LB basal medium (119 mg/L). This production rate is the highest hGH concentration reported in the IPTG-inducible expression systems.

  16. [Cloning and expression of a single human immunoglobulin heavy-chain variable domain with vascular endothelial growth factor binding activity].

    Science.gov (United States)

    Liu, Heng; Liu, Siguo; Wu, Yi; Zili, M; Liu, Yu; Zhang, Aimin; Chen, Jianquan; Cheng, Guoxiang

    2010-11-01

    In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.

  17. In vivo enhancement of chemosensitivity of human salivary gland cancer cells by overexpression of TGF-beta stimulated clone-22.

    Science.gov (United States)

    Omotehara, F; Uchida, D; Hino, S; Begum, N M; Yoshida, H; Sato, M; Kawamata, H

    2000-01-01

    We have isolated transforming growth factor-beta-stimulated clone-22 (TSC-22) cDNA as an anti-cancer drug-inducible gene in a human salivary gland cancer cell line, TYS. We have previously reported that TSC-22 negatively regulates the growth of TYS cells, and that overexpression of TSC-22 protein in TYS cells enhanced the in vitro chemosensitivity of the cells. In this study, we examined the in vivo chemosensitivity of TSC-22-expressing TYS cells. TSC-22-expressing TYS cells formed tumors in nude mice, but tumors formed by TSC-22-expressing TYS cells were significantly smaller than tumors formed by control cells (pway ANOVA). Furthermore, intraperitoneal injection of 5-fluorouracil (5-FU) markedly inhibited the growth of the TSC-22-expressing TYS tumors, but did not affect the growth of control tumors. It was found by TUNEL assay that TSC-22-expressing TYS tumors were induced to undergo apoptosis by 5-FU treatment. These findings suggest that overexpression of TSC-22 protein in TYS cells enhances the in vivo chemosensitivity of the cells to 5-FU via induction of apoptosis.

  18. Ethical issues in animal cloning.

    Science.gov (United States)

    Fiester, Autumn

    2005-01-01

    The issue of human reproductive cloning has recently received a great deal attention in public discourse. Bioethicists, policy makers, and the media have been quick to identify the key ethical issues involved in human reproductive cloning and to argue, almost unanimously, for an international ban on such attempts. Meanwhile, scientists have proceeded with extensive research agendas in the cloning of animals. Despite this research, there has been little public discussion of the ethical issues raised by animal cloning projects. Polling data show that the public is decidedly against the cloning of animals. To understand the public's reaction and fill the void of reasoned debate about the issue, we need to review the possible objections to animal cloning and assess the merits of the anti-animal cloning stance. Some objections to animal cloning (e.g., the impact of cloning on the population of unwanted animals) can be easily addressed, while others (e.g., the health of cloned animals) require more serious attention by the public and policy makers.

  19. cDNA cloning, chromosome mapping and expression characterization of human geranylgeranyl pyrophosphate synthase

    Institute of Scientific and Technical Information of China (English)

    赵勇[1; 余龙[2; 高洁[3; 付强[4; 华益民[5; 张宏来[6; 赵寿元[7

    2000-01-01

    Geranylgeranyl pyrophosphate (GGPP) mainly participates in post-translational modification for various proteins including Rho/Rac, Rap and Rab families, as well as in regulation for cell apoptosis. Geranylgeranyl pyrophosphate synthase (GGPPS), which catalyzes the condensation reaction between farnesyl diphosphate and isopentenyl diphosphate, is the key enzyme for synthesizing GGPP. We report the isolation of a gene transcript showing high homology with Drosophila GGPPS cDNA. The transcript is 1 466 bp in length and contains an intact open reading frame (ORF) ranging from nt 239 to 1 138. This ORF encodes a deduced protein of 300 residues with calculated molecular weight of 35 ku. The deduced protein shows 57.5% identity and 75% similarity with Drosophila GGPPS, and contains five characteristic domains of prenyltransferases. Northern hybridization revealed that human GGPPS was expressed highest in heart, and moderately in spleen, testis, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas

  20. Phage display-based strategies for cloning and optimization of monoclonal antibodies directed against human pathogens.

    Science.gov (United States)

    Clementi, Nicola; Mancini, Nicasio; Solforosi, Laura; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2012-01-01

    In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

  1. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 k......, respectively, hybridizing specifically with the β2gpI cDNA. Upon isoelectric focusing, recombinant β2gpI obtained from expression of β2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as β2gpI isolated from plasma, and at least 5 polypeptides were visible...

  2. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambdagt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

  3. Therapeutic and reproductive cloning: a critique.

    Science.gov (United States)

    Bowring, Finn

    2004-01-01

    This article is a critical examination of the science and ethics of human cloning. It summarises the key scientific milestones in the development of nuclear transplantation, explains the importance of cloning to research into the medical potential of embryonic stem cells, and discusses the well-worn distinction between 'therapeutic' and 'reproductive' cloning. Suggesting that this distinction will be impossible to police, it goes on to consider the ethics of full human cloning. It is concluded that it represents an unacceptable form of parental despotism, and that the genetic engineering and cloning of future human beings will fracture the foundations of modern humanism.

  4. Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

    Science.gov (United States)

    Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

    1985-12-01

    A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

  5. CLONING AND IDENTIFICATION OF A GENE RELATED TO THE DIFFERENTIATION OF HUMAN GASTRIC ADENOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    王建华; 陈诗书

    2002-01-01

    Objective: To compare the differential expression of mRNA between MKN-28 (highly differentiated) and MKN-45 (poorly differentiated) gastric adenocarcinoma cells and identify genes involved in human gastric adenocarcinoma differentiation. Methods: Differential expression of mRNA between MKN-28 and MKN-45 adenocarcinoma cells was investigated by fluorescent differential display (FDD). Differentially expressed cDNA was analyzed by bio-informatics and confirmed by RT-PCR and Northern-blot. Results: 45 differential fragments were finally attained. One of them (No. 10) was an approximate 750 bp cDNA and highly up-regulated in MKN-45 cells as compared with MKN-28 cells. By using Blastn and UniGene database analysis, we found the fragment was mapped to chromosome 14q11.2(q12 and showed a significant homology to Bcl-2 binding protein gene (BNip3), which was recently identified encoding pro-apoptosis protein located in mitochondrial. Conclusion: The BNip3 induced apoptosis could be suppressed by interacting with bcl-2. The BNip3 gene in tumor cells might be up-regulated by the hypoxia response element through the HIF1a transcription factor, causing death of the hypoxic cells at the center of the tumor where vascularization is usually poor in the process of tumor development.

  6. Phage Display-based Strategies for Cloning and Optimization of Monoclonal Antibodies Directed against Human Pathogens

    Directory of Open Access Journals (Sweden)

    Roberto Burioni

    2012-07-01

    Full Text Available In the last two decades, several phage display-selected monoclonal antibodies (mAbs have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.

  7. Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.

    Science.gov (United States)

    Jaye, M; de la Salle, H; Schamber, F; Balland, A; Kohli, V; Findeli, A; Tolstoshev, P; Lecocq, J P

    1983-04-25

    A unique 52mer oligonucleotide deduced from the amino acid sequence of bovine Factor IX was synthesized and used as a probe to screen a human liver cDNA bank. The Factor IX clone isolated shows 5 differences in nucleotide and deduced amino acid sequence as compared to a previously isolated clone. In addition, precisely one codon has been deleted.Images

  8. Metabolism of rutin and poncirin by human intestinal microbiota and cloning of their metabolizing α-L-rhamnosidase from Bifidobacterium dentium.

    Science.gov (United States)

    Bang, Seo-Hyeon; Hyun, Yang-Jin; Shim, Juwon; Hong, Sung-Woon; Kim, Dong-Hyun

    2015-01-01

    To understand the metabolism of flavonoid rhamnoglycosides by human intestinal microbiota, we measured the metabolic activity of rutin and poncirin (distributed in many functional foods and herbal medicine) by 100 human stool specimens. The average α-Lrhamnosidase activities on the p-nitrophenyl-α-L-rhamnopyranoside, rutin, and poncirin subtrates were 0.10 ± 0.07, 0.25 ± 0.08, and 0.15 ± 0.09 pmol/min/mg, respectively. To investigate the enzymatic properties, α-L-rhamnosidase-producing bacteria were isolated from the specimens, and the α-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned α-L-rhamnosidase gene contained a 2,673 bp sequcence encoding 890 amino acid residues. The cloned gene was expressed using the pET 26b(+) vector in E. coli BL21, and the expressed enzyme was purified using Ni(2+)-NTA and Q-HP column chromatography. The specific activity of the purified α-L-rhamnosidase was 23.3 μmol/min/mg. Of the tested natural product constituents, the cloned α-L-rhamnosidase hydrolyzed rutin most potently, followed by poncirin, naringin, and ginsenoside Re. However, it was unable to hydrolyze quercitrin. This is the first report describing the cloning, expression, and characterization of α-L-rhamnosidase, a flavonoid rhamnoglycosidemetabolizing enzyme, from bifidobacteria. Based on these findings, the α-L-rhamnosidase of intestinal bacteria such as B. dentium seem to be more effective in hydrolyzing (1-->6) bonds than (1-->2) bonds of rhamnoglycosides, and may play an important role in the metabolism and pharmacological effect of rhamnoglycosides.

  9. Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein.

    Science.gov (United States)

    Pogue-Geile, K; Geiser, J R; Shu, M; Miller, C; Wool, I G; Meisler, A I; Pipas, J M

    1991-08-01

    We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library. The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa. Increased levels of the S3 transcript were present in the tumors of all eight patients examined. Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma. Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps. These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

  10. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

    Energy Technology Data Exchange (ETDEWEB)

    Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J. (Wistar Inst. of Anatomy and Biology, Philadelphia, PA (USA))

    1990-09-01

    The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins.

  11. A pluripotent human stem-cell clone isolated from the TERA-2 teratocarcinoma line lacks antigens SSEA-3 and SSEA-4 in vitro, but expresses these antigens when grown as a xenograft tumor.

    Science.gov (United States)

    Andrews, P W; Damjanov, I; Simon, D; Dignazio, M

    1985-01-01

    Human embryonal carcinoma (EC) cells generally express the cell-surface, stage-specific embryonic antigens 3 and 4 (SSEA-3 and SSEA-4), the epitopes of which are defined by two monoclonal antibodies that recognize different portions of an extended globoseries oligosaccharide. To examine further the relationship between these epitopes and the human EC phenotype, we investigated the properties of two newly isolated clones from the human teratocarcinoma cell line, TERA-2. One clone expresses SSEA-3 and SSEA-4; the other does not. Nevertheless, these clones otherwise resemble one another, and based upon their morphology, their expression of other cell-surface antigens, and their ability to form xenograft tumors containing a variety of cell types, we conclude that both clones are composed of pluripotent human EC cells. When exposed to retinoic acid in vitro, neither clone differentiates as extensively as other clones that we have previously derived from TERA-2. These observations indicate heterogeneity among stem cells derived from a single human teratocarcinoma, and suggest that SSEA-3 and SSEA-4 are not necessarily integral features of the human EC phenotype. On the other hand, EC cells in xenograft tumors derived from the SSEA-3- and SSEA-4-negative clone re-express these epitopes. Further, this re-expression is stable, since EC cell lines that are SSEA-3- and SSEA-4-positive grow out when the tumors are explanted in vitro. We conclude that the expression of these globoseries epitopes can be modulated by environmental influences.

  12. Prostitution, disability and prohibition.

    Science.gov (United States)

    Thomsen, Frej Klem

    2015-06-01

    Criminalisation of prostitution, and minority rights for disabled persons, are important contemporary political issues. The article examines their intersection by analysing the conditions and arguments for making a legal exception for disabled persons to a general prohibition against purchasing sexual services. It explores the badness of prostitution, focusing on and discussing the argument that prostitution harms prostitutes, considers forms of regulation and the arguments for and against with emphasis on a liberty-based objection to prohibition, and finally presents and analyses three arguments for a legal exception, based on sexual rights, beneficence, and luck egalitarianism, respectively. It concludes that although the general case for and against criminalisation is complicated there is a good case for a legal exception.

  13. Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Rasmussen, H H

    1993-01-01

    We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from...

  14. Establishment of a transgenic cell line stably expressing human cytochrome P450 2C18 and identification of a CYP2C18 clone with exon 5 missing

    Institute of Scientific and Technical Information of China (English)

    Jian Zhu-Ge; Ying-Nian Yu; Yu-Li Qian; Xin Li

    2002-01-01

    AIM: The human cytochrome P-450 2C18(CYP2C18) hasbeen characterized. However, the protein has not beenpurified from liver and very little is known regarding thespecific substrate of CYP2C18. In order to study its enzymaticactivity for drug metabolism, the CYP2C18cDNA was clonedand a stable CHL cell line expressing recombinant CYP 2C18was established.METHODS: The human CYP2C18cDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR)from total RNAs extracted from human liver and cloned intopGEM-T vector. The cDNA segment was identified by DNAsequencing and subcloned into a mammalian expressionvector pREP9. A transgenic cell line was established bytransfecting the recombinant plasmid of pREPg-CYP2C18toChinese hamster lung (CHL) cell. The enzyme activity ofCYP2C18 catalyzing oxidation of tolbutamide tohydroxytolbutamide in postmitochondrial supernant(Sg)fraction of the cell was determined by high performanceliquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from thecloned cDNA segment was identical to that of reported byRomkes et al(GenBank accession number: M61856,J05326).The S9 fraction of the established cell line metabolizestolbutamide to hydroxytolbutamide. Tolbutamide hydroxylaseactivity was found to be 0.509±0.052 μmol.min-1.g-1 S9protein or 8.82±0.90 mol.min-1.mol-1 CYP, but wasundetectable in parental CHL cell. In addition, we haveidentified a CYP2C18cDNA clone with exon 5 missing.CONCLUSION: The cDNA of human CYP2C18 wassuccessfully cloned and a cell line, CHL-CYP2C18, efficientlyexpressing the protein of CYP2C18, was established. Aspliced variant of CYP2C18 with exon 5 missing was identifiedin the cloning process.

  15. 克隆人技术伦理根基之思考%Considerations on the Ethical Foundations of Human Cloning Technology

    Institute of Scientific and Technical Information of China (English)

    季爱民

    2014-01-01

    From the purpose of human cloning technology ,basic research of cloning generally does not cause a strong impact on the ethical issues ,while therapeutic and reproductive cloning really do .The ethical issues of reproductive cloning is especially detrimental to human dignity ,w hile the core of the ethical issues on therapeutic cloning is the rights and moral status of the embryonic .Taking the two property of truth and practice of human cloning technology into consideration ,as well as the relative criteria ,absolutely good or bad judge on the technology is not rigorous .However ,it is necessary to formulate ethical principles that regulate the technology field on bioethics .Among them ,the principle of informed consent is supposed to be an important one ,the ethics basis of w hich is :human being is ends ,not means .%从克隆人技术目的看,基础研究性克隆一般不会引起伦理上强烈冲击,治疗性和生殖性克隆则强烈冲击着人类伦理道德。生殖性克隆伦理问题的突出表现在于有损人类的尊严,治疗性克隆伦理问题的核心则是胚胎的权利与道德地位问题。考虑到克隆人技术的真理性和实践性两个层面,以及评判标准的相对性,对该技术进行绝对的善恶评判有失慎重。不过,生命伦理领域制定规范该技术的伦理原则是必须的,其中,知情同意原则理应是一个重要原则,其伦理学依据在于:人是目的,而不是手段。

  16. The Cloning of America.

    Science.gov (United States)

    Dobson, Judith E.; Dobson, Russell L.

    1981-01-01

    Proposes that the U.S. school system purports to prize human variability, but many educators are engaged in activities that seek to homogenize students. Describes these activities, including diagnosis, labeling, ability grouping, and positive reinforcement. Presents suggestions for counselors to combat sources of cloning and self-validation. (RC)

  17. Detection of cancer clones in human colorectal adenoma as revealed by increased DNA instability and other bio-markers

    Directory of Open Access Journals (Sweden)

    Y Jin

    2009-06-01

    Full Text Available An immunohistochemical differential staining of cancerous cells with anti-cytidine antibody after denaturation of nuclear DNA by acid hydrolysis with 2N HCl at 30°C for 20 min (DNA-instability test has been used as a marker for malignancy. The test was applied to bioptic tissues of human colorectal polyps assessed histopathologically as hyperplastic polyp (11 cases, tubular adenoma of mild (68 cases, moderate (102 cases, and severe (46 cases dysplasia, and adenocarcinoma (30 cases. The serial sections of the same tissues were also subjected to immunohistochemical staining for Ki67, p53, DNA-fragmentation factor 45 (DFF45 and vascular endothelial growth factor (VEGF. The DNA-instability test was positive in 30 (100% adenocarcinoma cases, 46 (100% severe dysplasia adenoma cases, 36 (35.29% moderate dysplasia adenoma cases, and 8 (11.76% mild dysplasia adenoma cases, indicating malignancy. All hyperplastic polyps were negative to the DNA-instability test. Furthermore, the percentage of glands positive in the DNAinstability test steadily increased in going from mild (10%, to moderate (35%, to severe (100% dysplasia, and adenocarcinoma (100%. All other biological markers tested in the present study showed significantly higher values in those adenoma glands that werepositive to the DNA-instability test, irrespective of the dysplasia grade, as compared to the markers in the adenoma glands that were negative to DNA instability testing. Furthermore, the former values were comparable to those in adenocarcinoma. The results indicate that cancer cell clones are already present at the adenoma stages showing positivity to DNA instability testing, enhanced proliferative activity, p53 mutation and induction of DFF45 and VEGF, at a time when the degree of morphological atypia are not yet large enough for them to be identified as cancer. These factors promote cancer cell proliferation, produce heterogeneous subclones due to DNA instability, enhance their

  18. 33 CFR 156.310 - Prohibited areas.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Prohibited areas. 156.310 Section... the Gulf of Mexico § 156.310 Prohibited areas. Lightering operations are prohibited within the following areas in the Gulf of Mexico: (a) Claypile—prohibited area. This prohibited area consists of...

  19. The topsy-turvy cloning law.

    Science.gov (United States)

    Brassington, Iain; Oultram, Stuart

    2011-03-01

    In debates about human cloning, a distinction is frequently drawn between therapeutic and reproductive uses of the technology. Naturally enough, this distinction influences the way that the law is framed. The general consensus is that therapeutic cloning is less morally problematic than reproductive cloning--one can hold this position while holding that both are morally unacceptable--and the law frequently leaves the way open for some cloning for the sake of research into new therapeutic techniques while banning it for reproductive purposes. We claim that the position adopted by the law has things the wrong way around: if we accept a moral distinction between therapeutic and reproductive cloning, there are actually more reasons to be morally worried about therapeutic cloning than about reproductive cloning. If cloning is the proper object of legal scrutiny, then, we ought to make sure that we are scrutinising the right kind of clone.

  20. Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3

    Energy Technology Data Exchange (ETDEWEB)

    Haiming Chen; Lalioti, M.D.; Perrin, G.; Antonarakis, S.E. [Univ. of Geneva Medical School (Switzerland)] [and others

    1996-07-01

    In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to 2 genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3{prime} UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown. 56 refs., 8 figs., 1 tab.

  1. Molecular cloning.

    Science.gov (United States)

    Lessard, Juliane C

    2013-01-01

    This protocol describes the basic steps involved in conventional plasmid-based cloning. The goals are to insert a DNA fragment of interest into a receiving vector plasmid, transform the plasmid into E. coli, recover the plasmid DNA, and check for correct insertion events.

  2. Development of transgenic cloned pig models of skin inflammation by DNA transposon-directed ectopic expression of human β1 and α2 integrin.

    Directory of Open Access Journals (Sweden)

    Nicklas Heine Staunstrup

    Full Text Available Integrins constitute a superfamily of transmembrane signaling receptors that play pivotal roles in cutaneous homeostasis by modulating cell growth and differentiation as well as inflammatory responses in the skin. Subrabasal expression of integrins α2 and/or β1 entails hyperproliferation and aberrant differentiation of keratinocytes and leads to dermal and epidermal influx of activated T-cells. The anatomical and physiological similarities between porcine and human skin make the pig a suitable model for human skin diseases. In efforts to generate a porcine model of cutaneous inflammation, we employed the Sleeping Beauty DNA transposon system for production of transgenic cloned Göttingen minipigs expressing human β1 or α2 integrin under the control of a promoter specific for subrabasal keratinocytes. Using pools of transgenic donor fibroblasts, cloning by somatic cell nuclear transfer was utilized to produce reconstructed embryos that were subsequently transferred to surrogate sows. The resulting pigs were all transgenic and harbored from one to six transgene integrants. Molecular analyses on skin biopsies and cultured keratinocytes showed ectopic expression of the human integrins and localization within the keratinocyte plasma membrane. Markers of perturbed skin homeostasis, including activation of the MAPK pathway, increased expression of the pro-inflammatory cytokine IL-1α, and enhanced expression of the transcription factor c-Fos, were identified in keratinocytes from β1 and α2 integrin-transgenic minipigs, suggesting the induction of a chronic inflammatory phenotype in the skin. Notably, cellular dysregulation obtained by overexpression of either β1 or α2 integrin occurred through different cellular signaling pathways. Our findings mark the creation of the first cloned pig models with molecular markers of skin inflammation. Despite the absence of an overt psoriatic phenotype, these animals may possess increased susceptibility to

  3. Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family

    DEFF Research Database (Denmark)

    Honoré, B; Leffers, H; Madsen, Peder

    1994-01-01

    We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa...

  4. Identification and cloning of a new category of DNA fragments which are poorly represented in human genomic libraries.

    Science.gov (United States)

    Wong, P; Myal, Y; Shui, R; Tenniswood, M

    1993-01-29

    We have developed an alternative strategy for the preparation of genomic libraries that ensures better representation of genomic sequences commonly underrepresented in genomic libraries constructed using standard protocols. To overcome the apparent bias against genomic sequences containing clusters of restriction sites we have used nonoptimized restriction digestions to generate a mixture of DNA fragments which have been cloned into the EMBL3 vector. To validate this protocol we have screened the EMBL3 library to identify a full length genomic clone of the prolactin-inducible gene (PIP). Screening 4 other, commercially available, genomic libraries prepared using standard protocols for restriction digestion of the genomic DNA failed to identify any full length clones. We show that this increase in the representation of the full length PIP gene in the EMBL3 genomic library is attributable to the method of insert preparation used and suggests that an additional subset of sequences that may be poorly represented in, or absent from, established libraries may be cloned using this modified protocol.

  5. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing.

    Science.gov (United States)

    Veres, Adrian; Gosis, Bridget S; Ding, Qiurong; Collins, Ryan; Ragavendran, Ashok; Brand, Harrison; Erdin, Serkan; Cowan, Chad A; Talkowski, Michael E; Musunuru, Kiran

    2014-07-03

    Genome editing has attracted wide interest for the generation of cellular models of disease using human pluripotent stem cells and other cell types. CRISPR-Cas systems and TALENs can target desired genomic sites with high efficiency in human cells, but recent publications have led to concern about the extent to which these tools may cause off-target mutagenic effects that could potentially confound disease-modeling studies. Using CRISPR-Cas9 and TALEN targeted human pluripotent stem cell clones, we performed whole-genome sequencing at high coverage in order to assess the degree of mutagenesis across the entire genome. In both types of clones, we found that off-target mutations attributable to the nucleases were very rare. From this analysis, we suggest that, although some cell types may be at risk for off-target mutations, the incidence of such effects in human pluripotent stem cells may be sufficiently low and thus not a significant concern for disease modeling and other applications.

  6. Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16S rRNA gene clone library analysis.

    Science.gov (United States)

    Li, Min; Zhou, Haokui; Hua, Weiying; Wang, Baohong; Wang, Shengyue; Zhao, Guoping; Li, Lanjuan; Zhao, Liping; Pang, Xiaoyan

    2009-05-01

    Bacteroides spp. represent a prominent bacterial group in human intestinal microbiota with roles in symbiosis and pathogenicity; however, the detailed composition of this group in human feces has yet to be comprehensively characterized. In this study, the molecular diversity of Bacteroides spp. in human fecal microbiota was analyzed from a seven-member, four-generation Chinese family using Bacteroides spp. group-specific 16S rRNA gene clone library analysis. A total of 549 partial 16S rRNA sequences amplified by Bacteroides spp.-specific primers were classified into 52 operational taxonomic units (OTUs) with a 99% sequence identity cut-off. Twenty-three OTUs, representing 83% of all clones, were related to 11 validly described Bacteroides species, dominated by Bacteroides coprocola, B. uniformis, and B. vulgatus. Most of the OTUs did not correspond to known species and represented hitherto uncharacterized bacteria. Relative to 16S rRNA gene universal libraries, the diversity of Bacteroides spp. detected by the group-specific libraries was much higher than previously described. Remarkable inter-individual differences were also observed in the composition of Bacteroides spp. in this family cohort. The comprehensive observation of molecular diversity of Bacteroides spp. provides new insights into potential contributions of various species in this group to human health and disease.

  7. Mitochondrial HMG to CoA synthase (mHS): cDNA cloning in human, mouse and C. elegans, mapping to human chromosome 1p12-13 and partial human genomic cloning

    Energy Technology Data Exchange (ETDEWEB)

    Boukaftane, Y.; Robert, M.F.; Mitchell, G.A. [Hopital Sainte-Justine, Montreal, Quebec (Canada)]|[Kingston General Hospital, Ontario (Canada)] [and others

    1994-09-01

    mHS catalyzes the rate-limiting first step of ketogenesis in the liver. A cytoplasmic HS isozyme, encoded by another gene, catalyzes an early step in cholesterol synthesis. Starting from a rat mHS cDNA obtained by RT-PCR from the published rat cDNA sequence, we obtained and sequenced human and mouse cDNAs spanning the entire coding sequence of natural human and mouse mHS, as well as sequencing C. elegans HS-like cDNA. Consensus sequences for 3 mitochondrial and 4 cytoplasmic HSs were created and compared to invertebrate HS sequences. We found high conversation in the active site and at other regions presumably important for HS function. We mapped the mHS locus, HMGCS2 by in situ hybridization to chromosome 1P12-13, in contrast to the human cHS locus (HMGCS1) known to be on chromosome 5p13. Comparative mapping results suggest that these two chromosomal regions may be contiguous in other species, constant with a recent gene duplication event. Furthermore, we have characterized a human genomic mHS subclone containing 4 mHS exons, and found the position of all splice junctions to be identical to that of the hamster cHS gene except for one site in the 3{prime} nontranslated region. We calculate that the mHS and cHS genes were derived from a common ancestor 400-700 Myrs ago, implying that ketogenesis from fat may have become possible around the time of emergence of vertebrates ({approximately}500 Myr ago). Ketogenesis has evolved into an important pathway of energy metabolism, and we predict the mHS deficiency may prove to be responsible for some as yet explained cases of Reye-like syndromes in humans. This hypothesis can now be tested at the molecular level without the necessity of obtaining hepatic tissue.

  8. The Budapest Meeting 2005 intensified networking on ethics of science: the case of reproductive cloning, germline gene therapy and human dignity.

    Science.gov (United States)

    Van Steendam, Guido; Dinnyés, András; Mallet, Jacques; Meloni, Rolando; Casabona, Carlos Romeo; González, Jorge Guerra; Kure, Josef; Szathmáry, Eörs; Vorstenbosch, Jan; Molnár, Péter; Edbrooke, David; Sándor, Judit; Oberfrank, Ferenc; Cole-Turner, Ron; Hargittai, István; Littig, Beate; Ladikas, Miltos; Mordini, Emilio; Roosendaal, Hans E; Salvi, Maurizio; Gulyás, Balázs; Malpede, Diana

    2006-10-01

    This paper reports on the meeting of the Sounding Board of the EU Reprogenetics Project that was held in Budapest, Hungary, 6-9 November 2005. The Reprogenetics Project runs from 2004 until 2007 and has a brief to study the ethical aspects of human reproductive cloning and germline gene therapy. Discussions during The Budapest Meeting are reported in depth in this paper as well as the initiatives to involve the participating groups and others in ongoing collaborations with the goal of forming an integrated network of European resources in the fields of ethics of science.

  9. A human Fab fragment specific for thyroid peroxidase generated by cloning thyroid lymphocyte-derived immunoglobulin genes in a bacteriophage lambda library.

    Science.gov (United States)

    Portolano, S; Seto, P; Chazenbalk, G D; Nagayama, Y; McLachlan, S M; Rapoport, B

    1991-08-30

    A human Fab fragment (SP2) which binds specifically to human thyroid peroxidase has been generated by expressing random combinations of heavy and light chain immunoglobulin genes (derived from Graves' thyroid cDNA) in a bacteriophage lambda library. In common with many serum TPO autoantibodies, the cloned Fab fragment is IgG1 kappa and has a high affinity for TPO (approximately 10(-9) M). On the basis of their nucleotide sequences, the heavy and light chain genes coding for SP2 belong to families VHI, (D), JH3 and VKI, JK2, respectively. These data provide the first characterization at a molecular level of a human thyroid peroxidase antibody associated with autoimmune thyroid disease.

  10. Entering the Clone Age

    Institute of Scientific and Technical Information of China (English)

    1995-01-01

    Suppose you make your parents so happy,they decide to have another baby just like you.It might be flattering,but how would you feel about having a little brother or sister who is also your twin? A laboratory experiment conducted last fall suggests it may someday be possible.For the first time ever,scientists made exact copies, or clones, of a human embryo.

  11. Therapeutic cloning in the mouse

    Science.gov (United States)

    Mombaerts, Peter

    2003-01-01

    Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of derivation of embryonic stem cell lines via nuclear transfer is remarkably consistent among these reports. However, the efficiency is so low that, in its present form, the concept is unlikely to become widespread in clinical practice. PMID:12949262

  12. Cloning: revisiting an old debate.

    Science.gov (United States)

    Verhey, Allen D

    1994-09-01

    The debate about cloning that took place 25 years ago, although directed toward a different sort of cloning, elucidates fundamental issues currently at stake in reproductive technologies and research. Paul Ramsey and Joseph Fletcher were participants in this early debate. The differences between Ramsey and Fletcher about the meaning and sufficiency of freedom, the understanding and weighing of good and evil, the connection between embodiment and personhood, the relationship of humans with nature, and the meaning of parenthood suggest both a broader agenda for the debate about cloning and a cautious move forward in the development of embryo-splitting.

  13. Cloning: Past, Present, and the Exciting Future. Breakthroughs in Bioscience.

    Science.gov (United States)

    Di Berardino, Marie A.

    This document explores the history of cloning by focusing on Dolly the Sheep, one of the first large animal clonings. The disadvantages and advantages of transgenic clones are discussed as well as the future implications of cloning from the perspective of human health. (Contains 10 resources.) (YDS)

  14. Gene cloning, expression and purification of human mito-chondrial tRNALeu(UUR) and its mutant

    Institute of Scientific and Technical Information of China (English)

    HAN; Weiguo

    2001-01-01

    [1]Anderson, S., Bankier, A. T., Barrell, B. G. et al., Sequence and organization of the human mitochondrial gene, Nature, 1981, 290(9): 457.[2]Wang, X. M., Yang, Y. S., The mutation of human mitochondrial tRNA and diseases, Chemistry of Life, 1999, 19(4): 171.[3]Goto, Y., Nonaka, I., Horai, S., A mutation in the tRNAleu(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1990, 348(13): 651.[4]Schapira, A. H. V., Mitochondrial disorders, Current Opinion in Genetics and Development, 1993, 3: 457.[5]Moraes, C. T., Ricci, E., Bonilla, W. et al., The mitochondrial tRNAleu(UUR) mutation in mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS): genetic, biochemical and morphological correlations in skeletal muscle, Am. J. Hum. Genet., 1992, 50: 934.[6]Chen, L., Jin, Y. X., Wang, D. B. et al., Species-specific identity elements of tRNATrp, Prog. Natl. Sci., 2000, 10(3): 192.[7]Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 556-558.[8]Merri, C. R., Goldman, D., Keuren, M. L. V., Gel protein stains: silver stain, Methods Enzymol, 1984, 104: 441.[9]Li, Y., Wang, E., Wang, Y., Overproduction and purification of Escherichia coli tRNAleu, Science in China, Ser. C, 1998, 41(3): 225.[10]Hess, J. F., Parisi, M. A., Bennett Clayton, D. A., Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies, Nature, 1991, 352: 236.[11]Yousufzai, A. Y. K., Bradford, M. W., Shrago, E. et al., Characterization of the adenine nucleotide translocase of pancreatic islet mitochondria, FEBS Lett., 1982, 137: 201.[12]Welsh, N., Paabo, S., Welsh, M., Decreased mitochondrial gene expression in isolated islet of rats injected neonatally with streptozotocin, Diabetologia, 1991, 34: 626.[13] Chen, L., Jin

  15. Molecular Cloning of a Novel cDNA From Mus Muscular BALB/c Mice Encoding Glycosyl Hydrolase Family 1: A Homolog of HumanLactase-Phlorizin Hydrolase

    Institute of Scientific and Technical Information of China (English)

    WEI HE; ZHEN-YU JI; CHENG-YU HUANG

    2006-01-01

    Objective To study the mechanism of lactose intolerance (LI) by cloning the mouse lactase cDNA and recombining a vector. Methods Total murine RNA was isolated from the small intestine of a 4-week-old BALB/c mouse (♂).Gene-specific primers were designed and synthesized according to the cDNA sequences of lactase-phlorizin hydrolase (LPH) in human, rat, and rabbit. A coding sequence (CDS) fragment was obtained using RT-PCR, and inserted into a clone vector pNEB-193, then the cDNA was sequenced and analyzed using bioinformatics. Results The cDNA from the BALB/c mouse with 912 bp encoding 303 amino acid residues. Analysis of the deduced amino acid sequence using bioinformatics revealed that this cDNA shared extensive sequence homology with human LPH containing a conserved glycosy1 hydrolase family 1 motif important for regulating lactase intolerance. Conclusion BALB/c mouse LPH cDNA (GenBank accession No: AY751548) provides a necessary foundation for study of the biological function and regulatory mechanism of the lactose intolerance in mice.

  16. 人源TNF-α抗体基因的克隆与表达%Cloning and expression of human TNF-α antibody gene

    Institute of Scientific and Technical Information of China (English)

    田露芳; 赵小玲; 张克军; 杨志华

    2009-01-01

    Objective: To express the TNF-α moneclonal antibody VH/K21 in the prokaryotic expression system, in order to further explore its biological functions and predict its clinical application prospect. Methods: The screened TNF-α an-tibedy VH/K21 gene was amplified by PCR and cloned into the prokaryotie expression vector pET-22b(+)/BL21(DE3), and achieved soluble expression. The products were identified by Western blotting and ELISA. Results: The soluble and high expressed human TNF-α monoclonal antibody was successfully cloned and expressed. Sequence analysis suggested that this clone had fully new human TNF-α antibody gene, and it could specifically recognize human TNF-α. Conclusion: The obtain of human TNF-α monoclonal antibody lays a foundation for its clinical application, and would provide a theoretical and technical basis for the preparation of other human antibodies.%目的:在原核系统中高效表达抗TNF-α单克隆抗体VH/K21,以便进一步研究其生物学功能,预测临床应用前景.方法:以已筛选获得的TNF-α抗体基因VH/K21为模板进行PCR扩增,产物与其表达质粒进行双酶切,连接后克隆进入PET22b(+)/BL21(DE3)感受态中并在该原核系统中实现可溶性表达;对表达产物进行Western blotting、ELISA鉴定.结果:成功克隆与表达出可溶性的高效表达的人源TNF-α单克隆抗体.序列分析表明该克隆具有全新的人源TNF-α抗体基因,它可以特异识别人TNF-α.结论:人源TNF-α单克隆抗体的获得为进一步研制应用于临床的TNF-α人源抗体奠定了基础,也将为其他人源抗体的制备提供理论依据和技术基础.

  17. Construction and characterization of a new simian/human immunodeficiency viruses clone carrying an env gene derived from a CRF07_BC strain

    Institute of Scientific and Technical Information of China (English)

    LI Yue; YANG Gui-bo; CHEN Qi-min; LIU Qiang; MENG Zhe-feng; GENG Yun-qi; QIAO Wen-tao; SHAO Yi-ming

    2009-01-01

    Background The CRF07_BC recombinant strain has been one of the most predominantly circulated HIV-1 strains in China, it is therefore necessary and urgent to develop a relevant animal model to evaluate candidate vaccines targeting HIV-1 CRF07_BC. A highly replication-competent simian/human immunodeficiency viruses (SHIV) construct containing the Chinese CRF07_BC HIV-1 env gene with the ability to infect Chinese rhesus monkeys would serve as an important tool in the development of HIV vaccines. The aim of this study was to examine whether SHIV XJDC6431 with the env fragment from a Chinese HIV-1 isolate virus could infect the human and monkey peripheral blood mononuclear cell (PBMC), establish infection in Chinese rhesus macaque.Methods A SHIV strain was constructed by replacing the rev/env genes of SHIV KB9 with the corresponding fragment derived from the HIV-1 CRF07_BC strain. The infectious activity of the SHIV clones was determined in vitro in PBMCs from both non-human primate animals and humans. Finally, one Chinese rhesus macaques (Macaca mulatto) was infected with one SHIV via intravenous infusion.Results One SHIV clone designated as SHIV XJDC6431, was generated that could infect macaque and human PBMC. The virus produced from this clone also efficiently infected the CCR5-expressing GHOST cell lines, indicating that it uses CCR5 as its coreceptor. Finally, the virus was intravenously inoculated into one Chinese rhesus macaque. Eventually, the animal became infected as shown by the occurrence of viremia within 3 of infection. The viral load reached 10~5 copies of viral RNA per ml of plasma during the acute phase of infection and lasted for 10 weeks post infection. Conclusions We conclude that SHIV XJDC6431 is an R5-tropic chimeric virus, which can establish infection not only in vitro but also in vivo in the Chinese rhesus macaque. Although the animal inoculated with SHIV XJDC6431 became infected without developing a pathologic phenotype, the virus efficiently

  18. Cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene from the Treacher Collins syndrome candidate region at 5q32-q33.1

    Energy Technology Data Exchange (ETDEWEB)

    Dixon, J.; Loftus, S.K.; Gladwin, A.J. [Univ. of Manchester (United Kingdom)] [and others

    1995-03-20

    Treacher Collins syndrome is an autosomal dominant disorder of craniofacial development, the features of which include conductive hearing loss and cleft palate. Previous studies have shown that the Treacher Collins syndrome locus is flanked by D5S519 and SPARC, and a yeast artificial chromosome contig encompassing this {open_quotes}critical region{close_quotes} has been completed. In the current investigation a cosmid containing D5S519 has been used to screen a human placental cDNA library. This has resulted in the cloning of the human heparan sulfate-N-deacetylase/N-sulfotransferase gene. Two different mRNA species that have identical protein coding sequences but that differ in the size and sequence of the 3{prime} untranslated regions (3{prime}UTR) have been identified. The smaller species has a 3{prime}UTR of 1035 bp, whereas that of the larger is 4878 bp. 24 refs., 3 figs.

  19. Cloning goes to the movies.

    Science.gov (United States)

    Cormick, Craig

    2006-10-01

    Public attitude research conducted by Biotechnology Australia shows that one of the major sources of information on human reproductive cloning is movies. Traditionally, understanding of new and emerging technologies has come through the mass media but human cloning, being so widely addressed through the popular culture of movies, is more effectively defined by Hollywood than the news media or science media. But how well are the science and social issues of cloning portrayed in box office hits such as The Island, Multiplicity, Star Wars: Attack of the Clones and Jurassic Park? These movies have enormous reach and undoubted influence, and are therefore worth analyzing in some detail. This study looks at 33 movies made between 1971 and 2005 that address human reproductive cloning, and it categorizes the films based on their genre and potential influence. Yet rather than simply rating the quality of the science portrayed, the study compares the key messages in these movies with public attitudes towards cloning, to examine the correlations.

  20. Identification, cloning and characterization of a novel 47 kDa murine PKA C subunit homologous to human and bovine Cβ2

    Directory of Open Access Journals (Sweden)

    Ørstavik Sigurd

    2006-08-01

    Full Text Available Abstract Background Two main genes encoding the catalytic subunits Cα and Cβ of cyclic AMP dependent protein kinase (PKA have been identified in all vertebrates examined. The murine, bovine and human Cβ genes encode several splice variants, including the splice variant Cβ2. In mouse Cβ2 has a relative molecular mass of 38 kDa and is only expressed in the brain. In human and bovine Cβ2 has a relative molecular mass of 47 kDa and is mainly expressed in lymphoid tissues. Results We identified a novel 47 kDa splice variant encoded by the mouse Cβ gene that is highly expressed in lymphoid cells. Cloning, expression, and production of a sequence-specific antiserum and characterization of PKA catalytic subunit activities demonstrated the 47 kDa protein to be a catalytically active murine homologue of human and bovine Cβ2. Based on the present results and the existence of a human brain-specifically expressed Cβ splice variant designated Cβ4 that is identical to the former mouse Cβ2 splice variant, the mouse splice variant has now been renamed mouse Cβ4. Conclusion Murine lymphoid tissues express a protein that is a homologue of human and bovine Cβ2. The murine Cβ gene encodes the splice variants Cβ1, Cβ2, Cβ3 and Cβ4, as is the case with the human Cβ gene.

  1. Cloning-free CRISPR

    Directory of Open Access Journals (Sweden)

    Mandana Arbab

    2015-11-01

    Full Text Available We present self-cloning CRISPR/Cas9 (scCRISPR, a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis.

  2. Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

    DEFF Research Database (Denmark)

    Röpke, M; Hald, J; Guldberg, Per

    1996-01-01

    A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant...

  3. Construction of a yeast artificial chromosome contig encompassing the human acidic fibroblast growth factor (FGF1) gene: Toward the cloning of the ANLL/MDS tumor-suppressor gene

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Ing-Ming; Gilmore, E.C.; Liu, Yang; Payson, R.A. (Ohio State Univ., Columbus, OH (United States))

    1994-02-01

    The region surrounding the human acidic fibroblast growth factor (FGF1) locus on chromosome 5q31 is of particular interest since it represents a critical region consistently lost in acute nonlymphocytic leukemia (ANLL) or myelodysplastic syndrome (MDS) patients who have a demonstrable deletion of the distal portion of the long arm of chromosome 5. It is proposed that an ANLL/MDS leukemia suppressor gene resides on 5q31. The authors have previously shown that the gene is most likely localized between FGF1 and PDGFRB/CSF1R loci. The region has also been linked to at least four other genetic diseases, Treacher Collins syndrome, diastrophic dysplasia, limb-girdle muscular dystrophy, and an autosomal dominant deafness, by linkage analysis. Here, they describe yeast artificial chromosomes (YAC) spanning 450 kb around the FGF1 gene. Six YAC clones were isolated from a human YAC library and their restriction enzyme maps were determined. The overlap of the clones with each other and with FGF1 cosmid and phage clones was characterized. Three of the YAC clones were found to contain the entire FGF1 gene, which spans more than 100 kb. Proximal and distal ends of several of these YAC clones were isolated for further overlap cloning. The proximal ends of both Y2 and Y4 were localized to previously isolated FGF1 DNA by sequence analysis. The distal ends of these two clones also hybridized to a human-hamster hybrid containing chromosome 5 as the only human genetic material. These results suggest that these YAC clones represent colinear DNA around the FGF1 locus. None of the YAC clones were found to contain the CD 14 and GRL genes, the closest known proximal and distal markers (relative to the centromere) to the FGF1 gene, respectively. This contig is useful for the overlap cloning of the 5q31 region and for reverse genetic strategies for the isolation of disease genes in the region. 46 refs., 7 figs., 5 tabs.

  4. 21 CFR 530.21 - Prohibitions for food-producing animals.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Prohibitions for food-producing animals. 530.21... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS EXTRALABEL DRUG USE IN ANIMALS Specific Provisions Relating to Extralabel Use of Animal and Human Drugs in Food-Producing Animals § 530.21 Prohibitions...

  5. Molecular cloning of human heat shock protein 27 and study of its protective effects on oxidative damage in rat cardiomyocte H9c2

    Institute of Scientific and Technical Information of China (English)

    Li Liu; Xiaojin Zhang; Surong Jiang; Xiang Gao; Guoxain Ding; Yunlin Cheng

    2005-01-01

    Objective: To clone human cardiac heat shock protein 27(HSP27) gene and to determine the effects of HSP27 on the oxidative stress in rat cardiomyocyte cell line H9c2. Methods: Full length of HSP27 cDNA which got by RT-PCR was constructed into pCDNA3.1 + . The recombinant was transfected into rat cardiomyocyte cell line H9c2 and the stable transfection cell line was selected by G418. Then we observe the effects of HSP27 over-expression on LDH release and apoptosis induced H2O2 in H9c2. Results:1000 μmol/L H2O2 in HSP27 over-expression group and wild type group were 0.396 ± 0.017 vs. 0.390 ± 0.009 ( p > 0.05 ), 0.437 ± 0.014 vs. 0.416 ± 0.015 ( P < 0.05), 0.471 ± 0.018 vs. 0.417 ± 0.009 ( P < 0.001), 0. 505 ± 0.030 vs. 0. 657 ± 0.022 ( P < 0.001),and wild type group were (10.693 ± 1.122) % vs. (4.027 ± 1.628 )% ( P < 0.01). Conclusion: We cloned and constructed human cardiac HSP27 gene successfully, and over-expression of human HSP27 could inhibit oxidative damage significantly in H9c2.

  6. Definition of an epitope on NS3 recognized by human CD4+ cytotoxic T lymphocyte clones cross-reactive for dengue virus types 2, 3, and 4.

    Science.gov (United States)

    Kurane, I; Zeng, L; Brinton, M A; Ennis, F A

    1998-01-20

    The role of dengue virus-specific serotype-cross-reactive T lymphocytes in recovery from and pathogenesis of dengue virus infections is not known. In the present paper, we have defined a dengue serotype-cross-reactive epitope recognized by two CD4+ CD8- cytotoxic T lymphocyte (CTL) clones, JK36 and JK46. These T cell clones were established from the peripheral blood T lymphocytes of a dengue-3-immune donor, using a limiting dilution method. JK36 and JK46 were cross-reactive for dengue virus types 2, 3, and 4, but not for type 1, and recognized the NS3 protein. The smallest synthetic peptide recognized by JK36 was an 8-amino acid peptide that contains amino acids (aa) 226 to 233 (VVAAEMEE) of NS3. The smallest peptide recognized by JK46 was an 11-amino acid peptide that contains aa 224 to 234 (TRVVAAEMEEA). HLA-DR15 was the restriction allele for recognition of these peptides by both JK36 and JK46. This is the first epitope to be defined that is recognized by human CD4+ CTL cross-reactive for dengue virus types 2, 3, and 4.

  7. Structural Mechanism Underpinning Cross-reactivity of a CD8+ T-cell Clone That Recognizes a Peptide Derived from Human Telomerase Reverse Transcriptase*

    Science.gov (United States)

    Cole, David K.; van den Berg, Hugo A.; Lloyd, Angharad; Crowther, Michael D.; Beck, Konrad; Ekeruche-Makinde, Julia; Miles, John J.; Bulek, Anna M.; Dolton, Garry; Schauenburg, Andrea J.; Wall, Aaron; Fuller, Anna; Clement, Mathew; Laugel, Bruno; Rizkallah, Pierre J.; Wooldridge, Linda; Sewell, Andrew K.

    2017-01-01

    T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection. PMID:27903649

  8. Over-expression of TSC-22 (TGF-beta stimulated clone-22) markedly enhances 5-fluorouracil-induced apoptosis in a human salivary gland cancer cell line.

    Science.gov (United States)

    Uchida, D; Kawamata, H; Omotehara, F; Miwa, Y; Hino, S; Begum, N M; Yoshida, H; Sato, M

    2000-06-01

    We have recently isolated TSC-22 (transforming growth factor-beta-stimulated clone-22) cDNA as an anticancer, drug-inducible (with vesnarinone) gene in a human salivary gland cancer cell line, TYS. We have also reported that TSC-22 negatively regulates the growth of TYS cells and that down-regulation of TSC-22 in TYS cells plays a major role in salivary gland tumorigenesis (Nakashiro et al, 1998). In this study, we transfected TYS cells with an expression vector encoding the TSC-22-GFP (green fluorescent protein) fusion protein, and we established TSC-22-GFP-expressing TYS cell clones. Next, we examined (a) the subcellular localization of the fusion protein, (b) the sensitivity of the transfectants to several anticancer drugs (5-fluorouracil, cis-diaminedichloroplatinum, peplomycin), and (c) induction of apoptotic cell death in the transfectants by 5-fluorouracil treatment. The TSC-22-GFP fusion protein was clearly localized to the cytoplasm, but not to the nucleus. Over-expression of the TSC-22-GFP fusion protein did not affect cell growth, but significantly increased the sensitivity of the cells to the anticancer drugs (p way ANOVA). Furthermore, over-expression of the TSC-22-GFP fusion protein markedly enhanced 5-fluorouracil-induced apoptosis. These findings suggest that over-expression of TSC-22-GFP protein in TYS cells enhances the chemosensitivity of the cells via induction of apoptosis.

  9. Antibody to a human DNA repair protein allows for cloning of a Drosophila cDNA that encodes an apurinic endonuclease.

    Science.gov (United States)

    Kelley, M R; Venugopal, S; Harless, J; Deutsch, W A

    1989-03-01

    The cDNA of a Drosophila DNA repair gene, AP3, was cloned by screening an embryonic lambda gt11 expression library with an antibody that was originally prepared against a purified human apurinic-apyrimidinic (AP) endonuclease. The 1.2-kilobase (kb) AP3 cDNA mapped to a region on the third chromosome where a number of mutagen-sensitive alleles were located. The cDNA clone yielded an in vitro translation product of 35,000 daltons, in agreement with the predicted size of the translation product of the only open reading frame of AP3, and identical to the molecular size of an AP endonuclease activity recovered following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Drosophila extracts. The C-terminal portion of the predicted protein contained regions of presumptive DNA-binding domains, while the DNA sequence at the amino end of AP3 showed similarity to the Escherichia coli recA gene. AP3 is expressed as an abundant 1.3-kb mRNA that is detected throughout the life cycle of Drosophila melanogaster. Another 3.5-kb mRNA also hybridized to the AP3 cDNA, but this species was restricted to the early stages of development.

  10. Whole genome comparison of donor and cloned dogs

    OpenAIRE

    Kim, Hak-Min; Cho, Yun Sung; Kim, Hyunmin; Jho, Sungwoong; Son, Bongjun; Choi, Joung Yoon; Kim, Sangsoo; Lee, Byeong Chun; Bhak, Jong; Jang, Goo

    2013-01-01

    Cloning is a process that produces genetically identical organisms. However, the genomic degree of genetic resemblance in clones needs to be determined. In this report, the genomes of a cloned dog and its donor were compared. Compared with a human monozygotic twin, the genome of the cloned dog showed little difference from the genome of the nuclear donor dog in terms of single nucleotide variations, chromosomal instability, and telomere lengths. These findings suggest that cloning by somatic ...

  11. High-dimensional quantum cloning and applications to quantum hacking.

    Science.gov (United States)

    Bouchard, Frédéric; Fickler, Robert; Boyd, Robert W; Karimi, Ebrahim

    2017-02-01

    Attempts at cloning a quantum system result in the introduction of imperfections in the state of the copies. This is a consequence of the no-cloning theorem, which is a fundamental law of quantum physics and the backbone of security for quantum communications. Although perfect copies are prohibited, a quantum state may be copied with maximal accuracy via various optimal cloning schemes. Optimal quantum cloning, which lies at the border of the physical limit imposed by the no-signaling theorem and the Heisenberg uncertainty principle, has been experimentally realized for low-dimensional photonic states. However, an increase in the dimensionality of quantum systems is greatly beneficial to quantum computation and communication protocols. Nonetheless, no experimental demonstration of optimal cloning machines has hitherto been shown for high-dimensional quantum systems. We perform optimal cloning of high-dimensional photonic states by means of the symmetrization method. We show the universality of our technique by conducting cloning of numerous arbitrary input states and fully characterize our cloning machine by performing quantum state tomography on cloned photons. In addition, a cloning attack on a Bennett and Brassard (BB84) quantum key distribution protocol is experimentally demonstrated to reveal the robustness of high-dimensional states in quantum cryptography.

  12. High-dimensional quantum cloning and applications to quantum hacking

    Science.gov (United States)

    Bouchard, Frédéric; Fickler, Robert; Boyd, Robert W.; Karimi, Ebrahim

    2017-01-01

    Attempts at cloning a quantum system result in the introduction of imperfections in the state of the copies. This is a consequence of the no-cloning theorem, which is a fundamental law of quantum physics and the backbone of security for quantum communications. Although perfect copies are prohibited, a quantum state may be copied with maximal accuracy via various optimal cloning schemes. Optimal quantum cloning, which lies at the border of the physical limit imposed by the no-signaling theorem and the Heisenberg uncertainty principle, has been experimentally realized for low-dimensional photonic states. However, an increase in the dimensionality of quantum systems is greatly beneficial to quantum computation and communication protocols. Nonetheless, no experimental demonstration of optimal cloning machines has hitherto been shown for high-dimensional quantum systems. We perform optimal cloning of high-dimensional photonic states by means of the symmetrization method. We show the universality of our technique by conducting cloning of numerous arbitrary input states and fully characterize our cloning machine by performing quantum state tomography on cloned photons. In addition, a cloning attack on a Bennett and Brassard (BB84) quantum key distribution protocol is experimentally demonstrated to reveal the robustness of high-dimensional states in quantum cryptography. PMID:28168219

  13. DNA cloning of human liver monoamine oxidase A and B: Molecular basis of differences in enzymatic properties

    Energy Technology Data Exchange (ETDEWEB)

    Back, A.W.J.; Lan, N.C.; Johnson, D.L.; Abell, C.W.; Bembenek, M.E.; Kwan, S.W.; Seeburg, P.H.; Shih, J.C. (Univ. of Heidelberg (West Germany))

    1988-07-01

    The monoamine oxidases play a vital role in the metabolism of biogenic amines in the central nervous system and in peripheral tissues. Using oligonucleotide probes derived from three sequenced peptide fragments, the authors have isolated cDNA clones that encode the A and B forms of monoamine oxidase and have determined the nucleotide sequences of these cDNAs. Comparison of the deduced amino acid sequences shows that the A and B forms have subunit molecular weights of 59,700 and 58,800, respectively, and have 70% sequence identity. Both sequences contain the pentapeptide Ser-Gly-Gly-Cys-Tyr, in which the obligatory cofactor FAD is covalently bound to cysteine. Based on differences in primary amino acid sequences and RNA gel blot analysis of mRNAs, the A and B forms of monoamine oxidase appear to be derived from separate genes.

  14. Morquio A syndrome: Cloning, sequence, and structure of the human N-acetylgalactosamine 6-sulfatase (GALNS) gene

    Energy Technology Data Exchange (ETDEWEB)

    Morris, C.P.; Guo, Xiao-Hui; Apostolou, S. [Adelaide Children`s Hospital, North Adelaide (Australia)] [and others

    1994-08-01

    Deficiency of the lysosomal enzyme, N-acetylgalactosamine 6-sulfatase (GALNS;EC 3.1.6.4), results in the storage of the glycosaminoglycans, keratan sulfate and chrondroitin 6-sulfate, which leads to the lysosomal storage disorder Morquio A syndrome. Four overlapping genomic clones derived from a chromosome 16-specific gridded cosmid library containing the entire GALNS gene were isolated. The structure of the gene and the sequence of the exon/intron boundaries and the 5{prime} promoter region were determined. The GALNS gene is split into 14 exons spanning approximately 40 kb. The potential promoter for GALNS lacks a TATA box but contains GC box consensus sequences, consistent with its role as a housekeeping gene. The GALNS gene contains an Alu repeat in intron 5 and a VNTR-like sequence in intron 6. 12 refs., 3 figs., 1 tab.

  15. cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

    DEFF Research Database (Denmark)

    Young, M F; Kerr, J M; Termine, J D

    1990-01-01

    A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

  16. Entangled Cloning of Stabilizer Codes and Free Fermions

    CERN Document Server

    Hsieh, Timothy H

    2016-01-01

    Though the no-cloning theorem prohibits exact replication of arbitrary quantum states, there are many instances in quantum information processing and entanglement measurement in which some generalization of a clone may be useful. Here, I provide a construction for generating an "entangled clone" for a particular but rather expansive and rich class of states. Given a stabilizer code or free fermion Hamiltonian, this construction generates an exact entangled clone of the original ground state, in the sense that the entanglement between the original and the exact copy can be tuned to be arbitrarily small but finite, or large, and the relation between the original and the copy can also be modified to some extent. For example, this work focuses on generating time-reversed copies of stabilizer codes and particle-hole transformed ground states of free fermion systems, although untransformed clones can also be generated. The protocol leverages entanglement to simulate a transformed copy of the Hamiltonian without hav...

  17. Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen

    Energy Technology Data Exchange (ETDEWEB)

    Federsppiel, B.; Melhado, I.G.; Delaney, A.; Clark-Lewis, I. (Univ. of British Columbia, Vancouver (Canada)); Duncan, A.M.V. (Queens Univ., Kinston, Ontario (Canada)); Jirik, F.R. (Hospital for Sick Children, Toronto, Ontario (Canada))

    1993-06-01

    A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E, was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin. 32 refs., 5 figs.

  18. Cloning and expressing a recombinant human tissue inhibitor of metalloproteinase-2 (TIMP-2)in methylotrophic yeast Pichia pastoris and its characterizations

    Institute of Scientific and Technical Information of China (English)

    YAN Xunyou; ZHAO Hongliang; ZHANG Weiguang; XUE Chong; LIU Zhimin

    2007-01-01

    To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2)cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris,we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using a standard chemical synthesis technique,which was composed of frequently used codons in the highly expressed Pichia pastoris genes.Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing.The verified gene of TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2.The plasmid was transformed into GS115 cells of the methylotrophic yeast,Pichia pastoris by electroporation,and we got the expression cell through phenotype selection and induction with methanol.Separation,purification,and bioactivity analysis of the expressed products were performed.

  19. Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1.

    Science.gov (United States)

    Kernstock, Stefan; Koch-Nolte, Friedrich; Mueller-Dieckmann, Jochen; Weiss, Manfred S; Mueller-Dieckmann, Christoph

    2009-05-01

    Human ADP-ribosylhydrolase 1 (hARH1, ADPRH) cleaves the glycosidic bond of ADP-ribose attached to an Arg residue of a protein. hARH1 has been cloned, expressed heterologously in Escherichia coli, purified and crystallized in complex with K(+) and ADP. The orthorhombic crystals contained one monomer per asymmetric unit, exhibited a solvent content of 43% and diffracted X-rays to a resolution of 1.9 A. A prerequisite for obtaining well diffracting crystals was the performance of X-ray fluorescence analysis on poorly diffracting apo hARH1 crystals, which revealed the presence of trace amounts of K(+) in the crystal. Adding K-ADP to the crystallization cocktail then resulted in a crystal of different morphology and with dramatically improved diffraction properties.

  20. The human peripheral benzodiazepine receptor gene: cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia.

    Science.gov (United States)

    Lin, D; Chang, Y J; Strauss, J F; Miller, W L

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondria to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, we have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5' untranslated segment. The 5' flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of "house-keeping" genes. The encoded PBR mRNA is alternately spliced into two forms: "authentic" PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [3H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease.

  1. Cloning of Humanα-defensin-1(HNP-1) Gene and Construction of Its Eukaryotic Expression Vector

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    1 IntroductionDefensins are small cationic antimicrobial peptides that function in the host's innate immune system. The human defensin family includes three small peptides from the azurophil granules of polymorphonuclear cells named human neutrophil peptide (HNP)-1, HNP-2, HNP-3, which consist 5%-7% of the protein of human neutrophil. HNP-4 is approximately one hundred times less abundant. They demonstrate antibacterial, antifungal and antiviral properties in vitro. HNPs are important component of nonoxidat...

  2. Italy Cloning Guru Says Babies on Their Way

    Institute of Scientific and Technical Information of China (English)

    Stephanie; Holmes; 孙海羽

    2002-01-01

    The Italian fertility expert whose avowed aim is to create the first human clone,said on Wednesday three women were pregnant with clones, but complained that thebabies would be viewed as freaks by a hostile society.

  3. Cloning and Expression of EBI3 and p28 Subunits of Human Interleukin 27 in E. coli

    Directory of Open Access Journals (Sweden)

    Ameneh Koochaki

    2015-07-01

    Full Text Available Background: Interleukin-27 is a heterodimeric cytokine belonging to IL-12 and IL-23 families, secreted  by Antigen Presenting Cells(APCs .The IL-27 is composed of 2 subunits: Epstein-Barr virus induced gene 3 (EBI3 and p28. IL-27 is an anti-inflammatory cytokine which has an inhibitory effect on Th17 population and suppress the IL-17 expression. It is suggested that IL-27 could be a potent drug candidate for treating auto immune diseases.Methods: The IL-27 subunit genes were constructed into plasmid vectors, they sub-cloned into pETDeut-1an expression vector in restriction sites of BamHI, SacI and NotI. Subsequently the induction was done by IPTG and the recombinant proteins were confirmed by SDS-PAGE and Western Blot analysis using anti His-tag antibody.Results: The 28kDa and 25kDa protein bands were observed on SDS-PAGE and finally confirmed by Western blot technique.

  4. Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* carbohydrate-binding protein of the human rotavirus strain Wa

    Energy Technology Data Exchange (ETDEWEB)

    Kraschnefski, Mark J.; Scott, Stacy A. [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia); Holloway, Gavan; Coulson, Barbara S.; Itzstein, Mark von [Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010 (Australia); Blanchard, Helen, E-mail: h.blanchard@griffith.edu.au [Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland 9726 (Australia)

    2005-11-01

    The carbohydrate-binding component (VP8*{sub 64–223}) of the human Wa rotavirus spike protein has been overexpressed in E. coli, purified and crystallized in two different crystal forms. X-ray diffraction data have been collected that have enabled determination of the Wa VP8*{sub 64–223} structure by molecular replacement. Rotaviruses exhibit host-specificity and the first crystallographic information on a rotavirus strain that infects humans is reported here. Recognition and attachment to host cells, leading to invasion and infection, is critically linked to the function of the outer capsid spike protein of the rotavirus particle. In some strains the VP8* component of the spike protein is implicated in recognition and binding of sialic-acid-containing cell-surface carbohydrates, thereby enabling infection by the virus. The cloning, expression, purification, crystallization and initial X-ray diffraction analysis of the VP8* core from human Wa rotavirus is reported. Two crystal forms (trigonal P3{sub 2}21 and monoclinic P2{sub 1}) have been obtained and X-ray diffraction data have been collected, enabling determination of the VP8*{sub 64–223} structure by molecular replacement.

  5. Structure and cell-specific expression of a cloned human retinol binding protein gene: the 5'-flanking region contains hepatoma specific transcriptional signals.

    Science.gov (United States)

    D'Onofrio, C; Colantuoni, V; Cortese, R

    1985-08-01

    Human plasma retinol binding protein (RBP) is coded by a single gene and is specifically synthesized in the liver. We have characterized a lambda clone, from a human DNA library, carrying the gene coding for plasma RBP. Southern blot analysis and DNA sequencing show that the gene is composed of six exons and five introns. Primer elongation and S1 mapping experiments allowed the definition of the initiation of transcription and the identification of the putative promoter. The 5'-flanking region of the RBP gene was fused upstream to the coding sequence of the bacterial enzyme chloramphenicol acetyl transferase (CAT): the chimeric gene was introduced, by calcium phosphate precipitation, into the human hepatoma cell line Hep G2 and into HeLa cells. Efficient expression of CAT was obtained only in Hep G2. Primer elongation analysis of the RNA extracted from transfected Hep G2 showed that initiation of transcription of the transfected chimeric gene occurs at a position identical to that of the natural gene. Transcriptional analysis of Bal31 deletions from the 3' end of the RBP 5'-flanking DNA allowed the identification of the RBP gene promoter.

  6. Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

    NARCIS (Netherlands)

    Meng, L.; Wan, Y.; Sun, Y.; Zhang, Y.; Wang, Z.; Song, Y.; Wang, F.

    2013-01-01

    Background - Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos.

  7. Cloning of observables

    OpenAIRE

    Ferraro, Alessandro; Galbiati, Matteo; Paris, Matteo G. A.

    2005-01-01

    We introduce the concept of cloning for classes of observables and classify cloning machines for qubit systems according to the number of parameters needed to describe the class under investigation. A no-cloning theorem for observables is derived and the connections between cloning of observables and joint measurements of noncommuting observables are elucidated. Relationships with cloning of states and non-demolition measurements are also analyzed.

  8. 41 CFR 60-300.21 - Prohibitions.

    Science.gov (United States)

    2010-07-01

    ... employee that the test purports to measure, rather than reflecting the impaired sensory, manual, or... Discrimination Prohibited § 60-300.21 Prohibitions. The term discrimination includes, but is not limited to, the... service medal veteran to the discrimination prohibited by this part. (2) Contractual or other...

  9. 41 CFR 60-250.21 - Prohibitions.

    Science.gov (United States)

    2010-07-01

    ... with a disability that impairs sensory, manual, or speaking skills, the test results accurately reflect... Discrimination Prohibited § 60-250.21 Prohibitions. The term “discrimination” includes, but is not limited to... other protected veteran to the discrimination prohibited by this part. (2) Contractual or...

  10. 30 CFR 57.5041 - Smoking prohibition.

    Science.gov (United States)

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Smoking prohibition. 57.5041 Section 57.5041..., Physical Agents, and Diesel Particulate Matter Radiation-Underground Only § 57.5041 Smoking prohibition. Smoking shall be prohibited in all areas of a mine where exposure records are required to be kept...

  11. 50 CFR 216.82 - Dogs prohibited.

    Science.gov (United States)

    2010-10-01

    ... 50 Wildlife and Fisheries 7 2010-10-01 2010-10-01 false Dogs prohibited. 216.82 Section 216.82... Pribilof Islands Administration § 216.82 Dogs prohibited. In order to prevent molestation of fur seal herds, the landing of any dogs at Pribilof Islands is prohibited....

  12. 24 CFR 891.675 - Prohibited facilities.

    Science.gov (United States)

    2010-04-01

    ... 24 Housing and Urban Development 4 2010-04-01 2010-04-01 false Prohibited facilities. 891.675... and Individuals-Section 162 Assistance § 891.675 Prohibited facilities. The requirements for prohibited facilities for 202/162 projects are provided in § 891.315, except that Section 202/162...

  13. Interest prohibition and financial product innovation

    NARCIS (Netherlands)

    J.A. Bergstra; C.A. Middelburg

    2011-01-01

    We give a rough sketch of the Judaic, Greek, Islamic and Christian positions in the matter of interest prohibition during the last few millennia and discuss the way in which interest prohibition is dealt with in Islamic finance, the problems with authority-based arguments for interest prohibition, a

  14. 40 CFR 791.105 - Prohibited acts.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 31 2010-07-01 2010-07-01 true Prohibited acts. 791.105 Section 791.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT (CONTINUED) DATA REIMBURSEMENT Prohibited Acts § 791.105 Prohibited acts. Failure to provide information...

  15. 13 CFR 113.3 - Discrimination prohibited.

    Science.gov (United States)

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Discrimination prohibited. 113.3... Provisions § 113.3 Discrimination prohibited. To the extent not covered or prohibited by part 112 of this... employment decisions shall be made in a manner which ensures that discrimination on the basis of handicap...

  16. 15 CFR 8b.11 - Discrimination prohibited.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Discrimination prohibited. 8b.11 Section 8b.11 Commerce and Foreign Trade Office of the Secretary of Commerce PROHIBITION OF DISCRIMINATION... Practices § 8b.11 Discrimination prohibited. (a) General. (1) No qualified handicapped individual shall, on...

  17. 15 CFR 8b.4 - Discrimination prohibited.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Discrimination prohibited. 8b.4 Section 8b.4 Commerce and Foreign Trade Office of the Secretary of Commerce PROHIBITION OF DISCRIMINATION... Provisions § 8b.4 Discrimination prohibited. (a) General. No qualified handicapped individual shall, on the...

  18. 15 CFR 8.4 - Discrimination prohibited.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Discrimination prohibited. 8.4 Section... General Provisions; Prohibitions: Nondiscrimination Clause; Applicability to Programs § 8.4 Discrimination... discrimination under, any program to which this part applies. (b) Specific discriminatory acts prohibited. (1) A...

  19. 15 CFR 8b.16 - Discrimination prohibited.

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false Discrimination prohibited. 8b.16 Section 8b.16 Commerce and Foreign Trade Office of the Secretary of Commerce PROHIBITION OF DISCRIMINATION... Accessibility § 8b.16 Discrimination prohibited. No qualified handicapped individual shall, because a recipient...

  20. 27 CFR 7.54 - Prohibited statements.

    Science.gov (United States)

    2010-04-01

    ... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Prohibited statements. 7.54 Section 7.54 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU... § 7.54 Prohibited statements. (a) General prohibition. An advertisement of malt beverages must...

  1. Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

    Science.gov (United States)

    Santhanam, R; Naz, R K

    2001-09-01

    A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

  2. Nuclear transfer technology in mammalian cloning.

    Science.gov (United States)

    Wolf, D P; Mitalipov, S; Norgren, R B

    2001-01-01

    The past several years have witnessed remarkable progress in mammalian cloning using nuclear transfer (NT). Until 1997 and the announcement of the successful cloning of sheep from adult mammary gland or fetal fibroblast cells, our working assumption was that cloning by NT could only be accomplished with relatively undifferentiated embryonic cells. Indeed, live offspring were first produced by NT over 15 years ago from totipotent, embryonic blastomeres derived from early cleavage-stage embryos. However, once begun, the progression to somatic cell cloning or NT employing differentiated cells as the source of donor nuclei was meteoric, initially involving differentiated embryonic cell cultures in sheep in 1996 and quickly thereafter, fetal or adult somatic cells in sheep, cow, mouse, goat, and pig. Several recent reviews provide a background for and discussion of these successes. Here we will focus on the potential uses of reproductive cloning along with recent activities in the field and a discussion concerning current interests in human reproductive and therapeutic cloning.

  3. Metabolomic phenotyping of a cloned pig model

    Directory of Open Access Journals (Sweden)

    Callesen Henrik

    2011-08-01

    Full Text Available Abstract Background Pigs are widely used as models for human physiological changes in intervention studies, because of the close resemblance between human and porcine physiology and the high degree of experimental control when using an animal model. Cloned animals have, in principle, identical genotypes and possibly also phenotypes and this offer an extra level of experimental control which could possibly make them a desirable tool for intervention studies. Therefore, in the present study, we address how phenotype and phenotypic variation is affected by cloning, through comparison of cloned pigs and normal outbred pigs. Results The metabolic phenotype of cloned pigs (n = 5 was for the first time elucidated by nuclear magnetic resonance (NMR-based metabolomic analysis of multiple bio-fluids including plasma, bile and urine. The metabolic phenotype of the cloned pigs was compared with normal outbred pigs (n = 6 by multivariate data analysis, which revealed differences in the metabolic phenotypes. Plasma lactate was higher for cloned vs control pigs, while multiple metabolites were altered in the bile. However a lower inter-individual variability for cloned pigs compared with control pigs could not be established. Conclusions From the present study we conclude that cloned and normal outbred pigs are phenotypically different. However, it cannot be concluded that the use of cloned animals will reduce the inter-individual variation in intervention studies, though this is based on a limited number of animals.

  4. Positional cloning of deafness genes

    NARCIS (Netherlands)

    Kremer, H.; Cremers, F.P.M.

    2009-01-01

    The identification of the majority of the known causative genes involved in nonsyndromic sensorineural hearing loss (NSHL) started with linkage analysis as part of a positional cloning procedure. The human and mouse genome projects in combination with technical developments on genotyping, transcript

  5. Cloning and identification of a novel RNF6 transcrip tional splice variant Spg2 in human development

    Institute of Scientific and Technical Information of China (English)

    ZHU Li; TONG GuoQin; CHEN Jie; WANG Yun; WANG SaiQun; ZHAO MuZhi; LI JianMin; MA JianHua

    2008-01-01

    RING finger proteins are zinc finger proteins containing the RING motifs. They act mainly as E3 ubiquitin ,gases, bind the ubiquitin E2 conjugating enzyme and promote degradation of targeted proteins,Many novel genes have been isolated and differentially expressed in human adult and embryo testis by a testis cDNA-array differential display technique. A novel RING finger cDNA is highly expressed in adult testis and at low level in fetal testis. It was named Spg2. It contains a 2055 nucleotide ORF, encodes a 685-amino-acid RNF6 protein, and has a RING finger in its C terminal. NCBI Blast shows that the gene is located on chromosome 13 and contains five exons. A multiple tissue expression profile also indicates that it is highly expressed in human testis, so we speculate that it may be associated with human spermatogenesis by virtue of the action of its RING domain.

  6. Cloning and identification of a novel RNF6 transcrip-tional splice variant Spg2 in human development

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    RING finger proteins are zinc finger proteins containing the RING motifs. They act mainly as E3 ubiq-uitin ligases, bind the ubiquitin E2 conjugating enzyme and promote degradation of targeted proteins, Many novel genes have been isolated and differentially expressed in human adult and embryo testis by a testis cDNA-array differential display technique. A novel RING finger cDNA is highly expressed in adult testis and at low level in fetal testis. It was named Spg2. It contains a 2055 nucleotide ORF, en-codes a 685-amino-acid RNF6 protein, and has a RING finger in its C terminal. NCBI Blast shows that the gene is located on chromosome 13 and contains five exons. A multiple tissue expression profile also indicates that it is highly expressed in human testis, so we speculate that it may be associated with human spermatogenesis by virtue of the action of its RING domain.

  7. Structure of the human zinc finger protein HIVEP3: molecular cloning, expression, exon-intron structure, and comparison with paralogous genes HIVEP1 and HIVEP2.

    Science.gov (United States)

    Hicar, M D; Liu, Y; Allen, C E; Wu, L C

    2001-01-01

    Here we report the cloning and characterization of HIVEP3, the newest member in the human immunodeficiency virus type 1 enhancer-binding protein family that encodes large zinc finger proteins and regulates transcription via the kappaB enhancer motif. The largest open reading frame of HIVEP3 contains 2406 aa. and is approximately 80% identical to the mouse counterpart. The HIVEP3 gene is located in the chromosomal region 1p34 and is at least 300 kb with 10 exons. RNA studies show that multiple HIVEP3 transcripts are differentially expressed and regulated. Additionally, transcription termination occurs in the ultimate exon, exon 10, or in exon 6. Therefore, HIVEP3 may produce protein isoforms that contain or exclude the carboxyl DNA binding domain and the leucine zipper by alternative RNA splicing and differential polyadenylation. Sequence homologous to HIVEP3 exon 6 is not found in mouse nor are the paralogous genes HIVEP1 and HIVEP2. Zoo-blot analysis suggests that sequences homologous to the human exon 6 are present only in primates and cow. Therefore, a foreign DNA harboring a termination exon likely was inserted into the HIVEP3 locus relatively recently in evolution, resulting in the acquisition of novel gene regulatory mechanisms as well as the generation of structural and functional diversity. Copyright 2001 Academic Press.

  8. Inhibition by a novel anti-arrhythmic agent, NIP-142, of cloned human cardiac K+ channel Kv1.5 current.

    Science.gov (United States)

    Matsuda, T; Masumiya, H; Tanaka, N; Yamashita, T; Tsuruzoe, N; Tanaka, Y; Tanaka, H; Shigenoba, K

    2001-03-16

    NIP-142 was shown to prolong atrial effective refractory period and to terminate atrial fibrillation and flutter in in vivo canine models. To obtain information on its antiarrhythmic action, we examined the effect of NIP-142 on cloned human cardiac K+ channel Kv1.5 (hKv1.5) currents stably expressed in a human cell line using whole-cell voltage clamp methods. NIP-142 inhibited the hKv1.5 current in a concentration-dependent and voltage-independent manner. The inhibition was larger at the end of depolarizing pulse than at the outward current peak. The IC50 for inhibition of the steady-state phase was 4.75 microM. A cross-over phenomenon was observed when current traces in the absence and presence of NIP-142 were superimposed. Inhibition of hKv1.5 current by NIP-142 was frequency-independent; changing the depolarizing pulse frequencies (0.1, 0.2, 1 Hz) and little effect on the degree of inhibition. NIP-142 decreased the maximal peak amplitude of kHv1.5 current at the first command pulse after 3 min rest in the presence of the drug. These results suggest that NIP-142 has inhibitory effects on the hKv 1.5 current through interaction with both open and closed states of the channel, which may underlie its antiarrhythmic activity in the atria.

  9. Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

    Science.gov (United States)

    Matsumura, Taku; Tatsumi, Kazuya; Noda, Yuichiro; Nakanishi, Naoyuki; Okonogi, Atsuhito; Hirano, Kunio; Li, Liu; Osumi, Takashi; Tada, Takashi; Kotera, Hidetoshi

    2014-10-10

    The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

  10. The human peripheral benzodiazepine receptor gene: Cloning and characterization of alternative splicing in normal tissues and in a patient with congenital lipoid adrenal hyperplasia

    Energy Technology Data Exchange (ETDEWEB)

    Lin, D.; Miller, W.L. (Univ. of California, San Francisco, CA (United States)); Chang, Y.J.; Strauss, J.F. III (Univ. of Pennsylvania School of Medicine, Philadelphia, PA (United States))

    1993-12-01

    The mitochondrial benzodiazepine receptor (mBzR) appears to be a key factor in the flow of cholesterol into mitochondia to permit the initiation of steroid hormone synthesis. The mBzR consists of three components; the 18-kDa component on the outer mitochondrial membrane appears to contain the benzodiazepine binding site, and is hence often termed the peripheral benzodiazepine receptor (PBR). Using a cloned human PBR cDNA as probe, the authors have cloned the human PBR gene. The 13-kb gene is divided into four exons, with exon 1 encoding only a short 5[prime] untranslated segment. The 5[prime] flanking DNA lacks TATA and CAAT boxes but contains a cluster of SP-1 binding sites, typical of [open quotes]housekeeping[close quotes] genes. The encoded PBR mRNA is alternately spliced into two forms: [open quotes]authentic[close quotes] PBR mRNA retains all four exons, while a short form termed PBR-S lacks exon 2. While PBR-S contains a 102-codon open reading frame with a typical initiator sequence, the reading frame differs from that of PBR, so that the encoded protein is unrelated to PBR. RT-PCR and RNase protection experiments confirm that both PBR and PBR-S are expressed in all tissues examined and that expression of PBR-S is about 10 times the level of PBR. Expression of PBR cDNA in pCMV5 vectors transfected into COS-1 cells resulted in increased binding of [[sup 3]H]PK11195, but expression of PBR-S did not. It has been speculated that patients with congenital lipoid adrenal hyperplasia, who cannot make any steroids, might have a genetic lesion in mBzR. RT-PCR analysis of testicular RNA from such a patient, sequencing of the cDNA, and blotting analysis of genomic DNA all indicate that the gene and mRNA for the PBR component of mBzR are normal in this disease. 36 refs., 6 figs.

  11. Antigen-bound C3b and C4b enhance antigen-presenting cell function in activation of human T-cell clones.

    Science.gov (United States)

    Arvieux, J; Yssel, H; Colomb, M G

    1988-10-01

    The effect of complement fragments C3b and C4b, on the triggering of antigen-specific human T-cell clones by Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) when these fragments are covalently coupled to the antigen tetanus toxin (TT) is described. TT was chemically cross-linked to purified C3b [(TT-C3b)n], C4b [(TT-C4b)n] or bovine serum albumin [(TT-BSA)n] as a control. T-cell activation was quantified by tritiated thymidine incorporation and 51Cr release. (TT-C3b)n and (TT-C4b)n induced proliferative responses comparable to (TT-BSA)n but at 18-25 and 4-6 lower concentrations, respectively. This enhancing effect required the covalent cross-linking of the complement fragments to the antigen and involved intracellular processing of the latter by LCL. Antigen presentation was similarly enhanced when measuring the cytotoxic activity of a helper T-cell clone against LCL previously pulsed with (TT-C3b)n or (TT-C4b)n compared with (TT-BSA)n. Binding studies, carried out on LCL using TT radiolabelled with 125I before cross-linking, indicated that (TT-C3b)n and (TT-C4b)n gave three- to four-fold more binding than (TT-BSA)n. Addition of antibodies against CR1 and CR2 or proteolytic removal of these complement receptors with trypsin inhibited by about 60% the enhancing effect of TT-bound C3b and C4b in both binding and functional assays. These results indicate that binding of C3b or C4b to antigen enhances antigen-specific proliferative and cytotoxic responses of T cells by targeting opsonized antigen onto complement receptors CR1 and CR2 of LCL. The putative significance of these findings in terms of regulation of immune responses by complement is discussed.

  12. Whole genome comparison of donor and cloned dogs.

    Science.gov (United States)

    Kim, Hak-Min; Cho, Yun Sung; Kim, Hyunmin; Jho, Sungwoong; Son, Bongjun; Choi, Joung Yoon; Kim, Sangsoo; Lee, Byeong Chun; Bhak, Jong; Jang, Goo

    2013-10-21

    Cloning is a process that produces genetically identical organisms. However, the genomic degree of genetic resemblance in clones needs to be determined. In this report, the genomes of a cloned dog and its donor were compared. Compared with a human monozygotic twin, the genome of the cloned dog showed little difference from the genome of the nuclear donor dog in terms of single nucleotide variations, chromosomal instability, and telomere lengths. These findings suggest that cloning by somatic cell nuclear transfer produced an almost identical genome. The whole genome sequence data of donor and cloned dogs can provide a resource for further investigations on epigenetic contributions in phenotypic differences.

  13. Molecular cloning and chromosomal localization of human genes encoding three closely related G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Zhao-Hui Song; Bonner, T.I. [NIMH National Inst. of Health, Bethesda, MD (United States); Modi, W. [Frederick Cancer Research and Development Center, Frederick, MD (United States)

    1995-07-20

    Cosmids containing human genes for orphan G protein-coupled receptors, GPR12, GPR6, and GPR3, were isolated using their rat homologs as probes. Previous studies of the mouse and rat cDNAs have shown the receptors to be expressed primarily in brain but have failed to identify their ligands. The three receptor proteins of 334, 363, and 330 amino acids, respectively, are encoded by a single exon in each gene. Excluding the divergent sequences preceding the first transmembrane domain, they have {approximately}60% amino acid identity with each other. Flurorescence in situ hybridization of GPR12, GPR6, and GPR3 localized these three genes to human chromosomal regions 13q12, 6q21, and 1p34.3-p36.1, respectively. 9 refs., 2 figs.

  14. Recovery of infectious human immunodeficiency virus type 1 after fusion of defectively infected clones of U-937 cells.

    OpenAIRE

    1991-01-01

    Polyethylene glycol was used to induce polykaryon formation among U-937 cell subclones carrying defective human immunodeficiency virus (HIV) type 1 proviral DNA. Fusion of cells which produced gp120-defective virions (UHC15.7) with cells unable to generate reverse transcriptase (RT) activity (UHC8 and UHC18) yielded polykaryons which made infectious viral progeny that showed normal protein profiles. Southern blot analysis of proviral DNA of cells infected with such fusion-derived virus reveal...

  15. Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils.

    Science.gov (United States)

    Smolen, J E; Hessler, R J; Nauseef, W M; Goedken, M; Joe, Y

    2001-08-01

    Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.

  16. Cloning of the VASP (Vasodilator-Stimulated Phosphoprotein) genes in human and mouse: Structure, sequence, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Zimmer, M.; Fischer, L.; Hauser, W. [Medizinische Universitaetsklinik, Wuerzburg (Germany)] [and others

    1996-09-01

    The genes encoding the vasodilator-stimulated phosphoprotein (VASP) in human and mouse were isolated, and major parts were sequenced. In both species the gene is composed of 13 exons with conserved exon-intron positions. The mouse VASP cDNA sequence was deduced from the genomic sequence. The predicted amino acid sequence is 89% identical to the human protein. The high nucleotide sequence homology extends not only over the coding regions but also into the 3{prime}-UTRs, indicating a possible function in mRNA targeting or regulation of translation. Prominent 5{prime} CpG islands including multiple SP1 sites indicate a housekeeping function of VASP. Using cosmid DNA as a probe for fluorescence in situ hybridization, the human VASP gene was assigned to chromosome 19q13.2-q13.3, an extended region with homology to mouse chromosome 7. A sequence overlap of the VASP 5{prime}-region with the telomeric end of a cosmid contig physically links the VASP gene with ERCC1. VASP is located about 92 kb distal to ERCC1 and about 300 kb proximal to the myotonic dystrophy protein kinase gene. 43 refs., 6 figs.

  17. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.D.; Funk, L.B.; Kennedy, M.E.; Pong, A.S.; Fitzgerald, G.A. (Vanderbilt Univ., Nashville, TN (United States))

    1991-06-01

    Platelets metabolize arachidonic acid to thromboxane A{sub 2}, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in the megakaryocytic/platelet-like HEL cell line. The PGG/H synthase gene was assigned to chromosome 9 by analysis of a human-hamster somatic hybrid DNA panel. The availability of platelet PGG/H synthase cDNA should enhance our understanding of the important structure/function domains of this protein and it gene regulation.

  18. Conflicting Rights: How the Prohibition of Human Trafficking and Sexual Exploitation Infringes the Right to Health of Female Sex Workers in Phnom Penh, Cambodia.

    Science.gov (United States)

    Maher, Lisa; Dixon, Thomas; Phlong, Pisith; Mooney-Somers, Julie; Stein, Ellen; Page, Kimberly

    2015-06-11

    While repressive laws and policies in relation to sex work have the potential to undermine HIV prevention efforts, empirical research on their interface has been lacking. In 2008, Cambodia introduced antitrafficking legislation ostensibly designed to suppress human trafficking and sexual exploitation. Based on empirical research with female sex workers, this article examines the impact of the new law on vulnerability to HIV and other adverse health outcomes. Following the introduction of the law, sex workers reported being displaced to streets and guesthouses, impacting their ability to negotiate safe sex and increasing exposure to violence. Disruption of peer networks and associated mobility also reduced access to outreach, condoms, and health care. Our results are consistent with a growing body of research which associates the violation of sex workers' human rights with adverse public health outcomes. Despite the successes of the last decade, Cambodia's AIDS epidemic remains volatile and the current legal environment has the potential to undermine prevention efforts by promoting stigma and discrimination, impeding prevention uptake and coverage, and increasing infections. Legal and policy responses which seek to protect the rights of the sexually exploited should not infringe the right to health of sex workers.

  19. The Limits of Pluralism - Recent Jurisprudence of the European Court of Human Rights with Regard to Minorities: Does the Prohibition of Discrimination Add Anything?

    Directory of Open Access Journals (Sweden)

    Sia Spiliopoulou Åkermark

    2002-10-01

    Full Text Available Since the mid-1990s, the European Court of Human Rights has had before it a number of cases concerning the situation of minorities under Article 14 of the European Convention for the Protection of Human Rights - which aims to secure the enjoyment of rights and freedoms without discrimination inter alia on grounds of association with a national minority. At present, the number of similar cases pending before the Court is growing. Through an examination of cases concerning mainly the nexus between Article 11 and Article 14 as well as Article 8 and Article 14, this article seeks to identify a number of problematic aspects of the jurisprudence of the Court. This, the author argues, includes uncertainty as to when and why the Court chooses to examine Article 14; issues of cumulative violations; issues of evidence; the questionable principle of prevention; issues of indirect discrimination and last, but not least, the potential benefits of the entry into force of Protocol No. 12. To address these problems, the author concludes that there is a need for greater coherency in the positions adopted by the Court with respect to minority issues as well as a need for more legal research.

  20. CLONING PROMOTER OF HUMAN SATB1 GENE AND EFFECT OF ATRA AND CoCl2 ON ITS ACTIVITY

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To explore the structure and activity of SATB1 promoter in different cells, ATRA and CoCl2 effect on its activity. Methods Using luciferase system to assay the promoter activity of human SATB1 gene, three luciferase reporter vectors were constructed which driven by different regions of 5' untranslated sequence from human SATB1 gene, called pGL3-SP2946-luc, pGL3-SP1718-luc and pGL3-SP751-luc, and transfected into Jurkat T, K562, U937 and Hela cells transiently using lipofectinamine, the expression activity was detected at different dosage of ATRA and CoCl2 treatment for different time course. Results The reporter gene expression from SATB1 promoter were high activity in U937 cell, moderate in Jurkat T cell, low activity in K562 cell and showed no obvious activity in Hela cell, the reporter gene expression from pGL3-SP751-luc kept on the higher lever in Jurkat T, K562 and U937 cells than the other two vectors. We also found that the repressive effect of CoCl2 on SATB1 's mRNA expression and the relative luciferase expression from pGL3-SP751-luc in U937 cell was down-regulated obviously by ATRA and CoCl2 in the concentration- and time-dependent manners. Conclusion SATB1 promoter drives gene expression with cell-specificity and its core promoter region maybe exist in the - 751 ~ - 9bp of 5' untranslated region of human SATB1 gene. Combined with the experiment result we found before that SATB1 was down-regulated by ATRA in U937, the results imply that STAB1 maybe is down-regulated by ATRA and CoCl2 through its promoter in the differentiation of myeloid cell line-U937.

  1. Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein.

    OpenAIRE

    Gray, P W; Barrett, K; Chantry, D; Turner, M.; Feldmann, M

    1990-01-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surf...

  2. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  3. Do Clones Dream of Love? Images of Clones in Popular Culture

    Directory of Open Access Journals (Sweden)

    Dragana Antonijević

    2016-02-01

    Full Text Available Fantasies about clones, cyborgs and androids have become part and parcel of the mythology of modern times – the mythologies of the biotechnological era in which the achievements of genetic engineering have inflamed fears of possible abuse of scientific knowledge and the consequences of such abuse. The paper considers the phenomenon of reproductive cloning of human beings as it is represented in popular culture, especially film as it is one of the most important sources of representations and constructions of ideas about clones. After the introductory consideration of this phenomenon in scientific, ethical and media debates which are imbued with rejection of reproductive cloning, I have analyzed the different uses of the clone motif in selected movies. I have examined the structure and content of the genre formula of "social melodrama" which is present in films about clones, and have analyzed the mythical patterns pertaining to the topic of cloning, such as the myth of immortality, the myth of twins, the myth of the uniqueness of human kind etc. Ultimately, the nature and origins of the fear of clones and disgust that clones cause have been examined, and it has been shown that they mostly boil down to the fear of the dehumanization of human beings, the fear of the loss of difference and the transgression of biological, sociocultural and metaphysical boundaries.

  4. Cloning non-transformed sheep B cells.

    Science.gov (United States)

    Griebel, P J; Beskorwayne, T; Godson, D L; Popowych, Y; Hein, W

    2000-04-03

    The capacity to clone B cells and establish permanent B cell lines has greatly facilitated a wide variety of studies characterising the growth, differentiation, and gene expression of murine and human B cells. Similar investigations of B cell biology for other species have been severely restricted by an inability to culture or clone B cells. This is the first report of a method to clone non-transformed sheep B cells using a culture system based on murine CD154 and a combination of human gamma chain-common cytokines. Sheep Peyer's patch B cells were cultured for 120 days and then cloned by limiting dilution culture. The parental B cell culture contained both surface immunoglobulin (sIg)M(+) and sIgG1(+) B cells and both types of B cell were cloned. Clonality was confirmed by PCR analysis of Ig heavy chain (HC) and light chain (LC) expression and DNA sequencing of HC V genes. There was agreement between the PCR and flow cytometric analyses of HC isotype expression on the B cell clones but the available monoclonal antibodies specific for sheep lambda and kappa LC did not react with all clones. Soluble Ig was detected in the culture supernatant of sIgG1(+) clones but not sIgM(+) clones. The B cell clones remained dependent upon CD154 and gamma chain-common cytokine co-stimulation for sustained growth and maintained stable Ig expression. The cloning of non-transformed sheep B cells should provide a valuable tool for studying sheep B cell biology, establishing Ig HC- and LC-specific monoclonal antibodies, analysing the B cell Ig repertoire, and may be used to produce sheep monoclonal antibodies.

  5. Cloning and characterization of CLCN5, the human kidney chloride channel gene implicated in Dent disease (an X-linked hereditary nephrolithiasis)

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, S.E.; Van Bakel, I.; Craig, I.W. [Univ. of Oxford (United Kingdom)] [and others

    1995-10-10

    Dent disease, an X-linked familial renal tubular disorder, is a form of Fanconi syndrome associated with proteinuria, hypercalciuria, nephrocalcinosis, kidney stones, and eventual renal failure. We have previously used positional cloning to identify the 3{prime} part of a novel kidney-specific gene (initially termed hClC-K2, but now referred to as CLCN5), which is deleted in patients from one pedigree segregating Dent disease. Mutations that disrupt this gene have been identified in other patients with this disorder. Here we describe the isolation and characterization of the complete open reading frame of the human CLCN5 gene, which is predicted to encode a protein of 746 amino acids, with significant homology to all known members of the ClC family of voltage-gated chloride channels. CLCN5 belongs to a distinct branch of this family, which also includes the recently identified genes CLCN3 and CLCN4. We have shown that the coding region of CLCN5 is organized into 12 exons, spanning 25-30 kb of genomic DNA, and have determined the sequence of each exon-intron boundary. The elucidation of the coding sequence and exon-intron organization of CLCN5 will both expedite the evaluation of structure/function relationships of these ion channels and facilitate the screening of other patients with renal tubular dysfunction for mutations at this locus. 31 refs., 5 figs.

  6. Interspecies spread of Staphylococcus aureus clones among companion animals and human close contacts in a veterinary teaching hospital. A cross-sectional study in Greece.

    Science.gov (United States)

    Drougka, Eleanna; Foka, Antigoni; Koutinas, Christos K; Jelastopulu, Eleni; Giormezis, Nikolaos; Farmaki, Ourania; Sarrou, Styliani; Anastassiou, Evangelos D; Petinaki, Efthimia; Spiliopoulou, Iris

    2016-04-01

    Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) prevalence among companion animals and veterinary personnel (VP) was investigated. Strains' molecular characteristics were evaluated in order to assess S. aureus transmission. Specimens (224) from colonized and infected sites of 102 animals (92 dogs, 10 cats) and 18 VP were collected during 2012 and 2013. Antibiotic susceptibility was performed by the disk diffusion method and Etest. mecA, mecC, tst (toxic shock syndrome toxin) and lukF/lukS-PV (Panton-Valentine leukocidin, PVL) genes were investigated by PCR. Genotypes were identified by Multi Locus Sequence Typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), accessory gene regulator group (agr), spa and Pulsed Field Gel Electrophoresis (PFGE). S. aureus prevalence among pets and VP was 36.3% (37/102) and 38.9% (7/18), respectively. Younger companion animals, those living in rural areas, having a disease upon admission or Coagulase-negative staphylococci co-carriage showed significantly higher prevalence of S. aureus isolation (panimals and VP. Companion animals harbor PVL-positive clones constituting a possible source for transmission to humans.

  7. Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong Li; You-Yong Lu

    2002-01-01

    AIM: To develop and optimize cDNA representationaldifference analysis (cDNA RDA) method and to identify andclone garlic up-regulated genes in human gastric cancer(HGC) cells.METHODS: We performed cDNA RDA method by usingabundant double-stranded cDNA messages provided by twoself-constructed cDNA libraries (Allitridi-trested and paternalHGC cell line BGC823 cells cDNA libraries respectively).BamH Ⅰ and Xho I restriction sites harbored in the libraryvector were used to select representations. Northern andSlot blots analyses were employed to identify the obtaineddifference products.RESJLTS: Fragments released from the cDNA library vectorafter restriction endonuclease digestion acted as goodmarker indicating the appropriate digestion degree for libraryDNA. Two novel expressed sequence tags (ESTs) and arecombinant gene were obtained. Slot blots result showed a8-fold increase of gila-derived nexin/protease nexin 1 (GDN/PN1 ) gene expression level and 4-fold increase of hepatitis Bvirus x-interacting protein (XIP) mRNA level in BGC823 cellsafter Allitridi treatment for 72 h.CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAsinduced by Allitridi provide valuable molecular evidence forelucidating the garlic' s efficacies against neurodegenerativeand inflammatory diseases. Isolation of a recombinant geneand two novel ESTs further show cDNA RDA based on cDNAlibraries to be a powerful method with high specificity andreproducibility in cloning differentially expressed genes.

  8. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of CofB, the minor pilin subunit of CFA/III from human enterotoxigenic Escherichia coli.

    Science.gov (United States)

    Kawahara, Kazuki; Oki, Hiroya; Fukakusa, Shunsuke; Maruno, Takahiro; Kobayashi, Yuji; Motooka, Daisuke; Taniguchi, Tooru; Honda, Takeshi; Iida, Tetsuya; Nakamura, Shota; Ohkubo, Tadayasu

    2015-06-01

    Colonization factor antigen III (CFA/III) is one of the virulence factors of human enterotoxigenic Escherichia coli (ETEC) that forms the long, thin, proteinaceous fibres of type IV pili through assembly of its major and minor subunits CofA and CofB, respectively. The crystal structure of CofA has recently been reported; however, the lack of structural information for CofB, the largest among the known type IV pilin subunits, hampers a comprehensive understanding of CFA/III pili. In this study, constructs of wild-type CofB with an N-terminal truncation and the corresponding SeMet derivative were cloned, expressed, purified and crystallized. The crystals belonged to the rhombohedral space group R32, with unit-cell parameters a = b = 103.97, c = 364.57 Å for the wild-type construct and a = b = 103.47, c = 362.08 Å for the SeMet-derivatized form. Although the diffraction quality of these crystals was initially very poor, dehydration of the crystals substantially improved the resolution limit from ∼ 4.0 to ∼ 2.0 Å. The initial phase was solved by the single-wavelength anomalous dispersion (SAD) method using a dehydrated SeMet CofB crystal, which resulted in an interpretable electron-density map.

  9. Cloning and expression in Escherichia coli of a human gelatinase B-inhibitory single-chain immunoglobulin variable fragment (scFv).

    Science.gov (United States)

    Zhou, N; Paemen, L; Opdenakker, G; Froyen, G

    1997-09-15

    The murine monoclonal antibody REGA-3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR-mediated cloning and sequenced. Single-chain Fv expression constructs for Escherichia coli were generated in which c-myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c-myc tag sequence was positioned at the amino-terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin-clipped F(ab')2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.

  10. Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

    Science.gov (United States)

    Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

    2014-10-01

    A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

  11. Molecular cloning and characterisation of a novel type of human papillomavirus 160 isolated from a flat wart of an immunocompetent patient.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Mitsuishi

    Full Text Available More than 150 types of Human papillomaviruses (HPVs have been isolated from numerous cutaneous and/or mucosal lesions. Flat wart samples on the face from 36 immunocompetent patients were collected and screened for HPV. From one sample, we cloned a putative novel genotype. The novel type consisted of 7779 bp in length with a GC content of 47.1%, containing open reading frames for putative early proteins (E1, E2, E4, E6, and E7 and two late proteins (L1 and L2. Homology searches and phylogenetic analyses indicated that it belonged to Alphapapillomavirus (Alpha-PV species 2 and most closely resembled HPV 3. The virus fulfilled the definition of a novel type, and was named HPV 160 by the Reference Center for Papillomaviruses. The putative E7 protein of HPV 160 as well as HPV 29, 77, and 78 contained the Leu-X-Cys-X-Glu pRB-binding motif but other Alpha-PV species 2 (HPV 3, 10, 28, 94, 117, and 125 did not have this conserved motif.

  12. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    Science.gov (United States)

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  13. Cloning and characterisation of hAps1 and hAps2, human diadenosine polyphosphate-metabolising Nudix hydrolases

    Directory of Open Access Journals (Sweden)

    Safrany Stephen T

    2002-07-01

    Full Text Available Abstract Background The human genome contains at least 18 genes for Nudix hydrolase enzymes. Many have similar functions to one another. In order to understand their roles in cell physiology, these proteins must be characterised. Results We have characterised two novel human gene products, hAps1, encoded by the NUDT11 gene, and hAps2, encoded by the NUDT10 gene. These cytoplasmic proteins are members of the DIPP subfamily of Nudix hydrolases, and differ from each other by a single amino acid. Both metabolise diadenosine-polyphosphates and, weakly, diphosphoinositol polyphosphates. An apparent polymorphism of hAps1 has also been identified, which leads to the point mutation S39N. This has also been characterised. The favoured nucleotides were diadenosine 5',5"'-pentaphosphate (kcat/Km = 11, 8 and 16 × 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2 and diadenosine 5',5"'-hexaphosphate (kcat/Km = 13, 14 and 11 × 103M-1s-1 respectively for hAps1, hAps1-39N and hAps2. Both hAps1 and hAps2 had pH optima of 8.5 and an absolute requirement for divalent cations, with manganese (II being favoured. Magnesium was not able to activate the enzymes. Therefore, these enzymes could be acutely regulated by manganese fluxes within the cell. Conclusions Recent gene duplication has generated the two Nudix genes, NUDT11 and NUDT10. We have characterised their gene products as the closely related Nudix hydrolases, hAps1 and hAps2. These two gene products complement the activity of previously described members of the DIPP family, and reinforce the concept that Ap5A and Ap6A act as intracellular messengers.

  14. Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families

    Energy Technology Data Exchange (ETDEWEB)

    Suchi, Mariko; Mizuno, Haruo; Tsuboi, Takashi [Nagoya City Univ. Medical School (Japan)] [and others

    1997-03-01

    Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5{prime}-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a {lambda}EMBL-3 human genomic library and report a single-copy gene spanning {approximately}15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5{prime} flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A- to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A ({nu} = .26) and 440 Gpoly ({nu} = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function. 76 refs., 5 figs., 7 tabs.

  15. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Spicer, E.K.; Horton, R.; Bloem, L.; Bach, R.; Williams, K.R.; Guha, A.; Kraus, J.; Lin, T.C.; Nemerson, Y.; Konigsberg, W.H.

    1987-08-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. lambda Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC).

  16. 45 CFR 73.735-803 - Prohibition against involvement in financial transactions based on information obtained through...

    Science.gov (United States)

    2010-10-01

    ... Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION STANDARDS OF CONDUCT Financial Interest § 73.735-803 Prohibition against involvement in financial transactions based on information... 45 Public Welfare 1 2010-10-01 2010-10-01 false Prohibition against involvement in financial...

  17. Update on the First Cloned Dog and Outlook for Canine Cloning.

    Science.gov (United States)

    Jang, Goo; Lee, ByeongChun

    2015-10-01

    As man's best friend, dogs have an important position in human society. Ten years ago, we reported the first cloned dog, and his birth has raised various scientific issues, such as those related to health, reproduction, and life span. He has developed without any unique health issues. In this article, we summarize and present perspectives on canine cloning.

  18. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the [alpha]1 (XVIII) collagen gene to mouse chromosome 10 and human chromosome 21

    Energy Technology Data Exchange (ETDEWEB)

    Oh, S.P.; Warman, M.L.; Timmons, S.; Olsen, B.R.; Knoll, J.H.M. (Harvard Medical School, Boston, MA (United States)); Seldin, M.F. (Duke Univ. Medical Center, Durham, NC (United States)); Cheng, Sou-De (Children' s Hospital/Harvard Medical School, Boston, MA (United States))

    1994-02-01

    Types XV and XVIII collagen belong to a unique and novel subclass of the collagen superfamily for which the authors have proposed the name the MULTIPLEXIN family. Members of this class contain polypeptides with multiple triple-helical domains separated and flanked by non-triple-helical regions. In this paper, they report the isolation of human cDNAs and genomic DNAs encoding the [alpha]1 (XVIII) collagen chain. Utilizing a genomic clone as probe, they have mapped the COL18A1 gene to chromosome 21q22.3 by fluorescence in situ hybridization. In addition, using an interspecific backcross panel, they have shown that the murine Col18a1 locus is on chromosome 10, close to the loci for Col6a1 and Col6a2. 16 refs., 5 figs.

  19. Quick and clean cloning.

    Science.gov (United States)

    Thieme, Frank; Marillonnet, Sylvestre

    2014-01-01

    Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

  20. A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

    OpenAIRE

    Liu Yi; Li Shangze; Zhang Huihui; Wan Zurong; Zhang Xiaodong; Du Runlei

    2012-01-01

    Abstract Background Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO) vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. Results We have developed a one-step cloning method for the insertion of two ...

  1. 50 CFR 401.4 - Activities prohibited.

    Science.gov (United States)

    2010-10-01

    ..., DEVELOPMENT AND ENHANCEMENT § 401.4 Activities prohibited. Law enforcement, public relations, harvesting, marketing and processing activities, construction of fisherman use facilities, and activities concerned...

  2. Deoxyhypusine hydroxylase from Plasmodium vivax, the neglected human malaria parasite: molecular cloning, expression and specific inhibition by the 5-LOX inhibitor zileuton.

    Directory of Open Access Journals (Sweden)

    Veronika Anyigoh Atemnkeng

    Full Text Available Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX. Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine(673. Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme.

  3. Cloning of the human type XVII collagen gene (COL17A1), and detection of novel mutations in generalized atrophic benign epidermolysis bullosa

    Energy Technology Data Exchange (ETDEWEB)

    Gatalica, B.; Pulkkinen, L.; Li, K. [Thomas Jefferson Univ., Philadelphia, PA (United States)] [and others

    1997-02-01

    Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal variant of junctional epidermolysis bullosa (JEB). Previous findings have suggested that type XVII collagen is the candidate gene for mutations in this disease. We now have cloned the entire human type XVII collagen gene (COL17A1) and have elucidated its intron-exon organization. The gene comprises 56 distinct exons, which span {approximately}52 kb of the genome, on the long arm of chromosome 10. It encodes a polypeptide, the {alpha}1(XVII) chain, consisting of an intracellular globular domain, a transmembrane segment, and an extracellular domain that contains 15 separate collagenous subdomains, the largest consisting of 242 amino acids. We also have developed a strategy to identify mutations in COL17A1 by use of PCR amplification of genomic DNA, using primers placed on the flanking introns. The PCR products are scanned for sequence variants by heteroduplex analysis using conformation-sensitive gel electrophoresis and then are subjected to direct automated sequencing. We have identified several intragenic polymorphisms in COL17A1, as well as mutations, in both alleles, in two Finnish families with GABEB. The probands in both families showed negative immunofluorescence staining with an anti-type XVII collagen antibody. In one family, the proband was homozygous for a 5-bp deletion, 2944del5, which resulted in frameshift and a premature termination codon of translation. The proband in the other family was a compound heterozygote, with one allele containing the 2944del5 mutation and the other containing a nonsense mutation, Q1023X. These results expand the mutation database in different variants of JEB, and they attest to the functional importance of type XVII collagen as a transmembrane component of the hemidesmosomes at the dermal/epidermal junction. 48 refs., 9 figs., 3 tabs.

  4. Cloning of the first human anti-JCPyV/VP1 neutralizing monoclonal antibody: epitope definition and implications in risk stratification of patients under natalizumab therapy.

    Science.gov (United States)

    Diotti, Roberta Antonia; Mancini, Nicasio; Clementi, Nicola; Sautto, Giuseppe; Moreno, Guisella Janett; Criscuolo, Elena; Cappelletti, Francesca; Man, Petr; Forest, Eric; Remy, Louise; Giannecchini, Simone; Clementi, Massimo; Burioni, Roberto

    2014-08-01

    JC virus (JCPyV) has gained novel clinical importance as cause of progressive multifocal leukoencephalopathy (PML), a rare demyelinating disease recently associated to immunomodulatory drugs, such as natalizumab used in multiple sclerosis (MS) cases. Little is known about the mechanisms leading to PML, and this makes the need of PML risk stratification among natalizumab-treated patients very compelling. Clinical and laboratory-based risk-stratification markers have been proposed, one of these is represented by the JCPyV-seropositive status, which includes about 54% of MS patients. We recently proposed to investigate the possible protective role of neutralizing humoral immune response in preventing JCPyV reactivation. In this proof-of-concept study, by cloning the first human monoclonal antibody (GRE1) directed against a neutralizing epitope on JCPyV/VP1, we optimized a robust anti-JCPyV neutralization assay. This allowed us to evaluate the neutralizing activity in JCPyV-positive sera from MS patients, demonstrating the lack of correlation between the level of anti-JCPyV antibody and anti-JCPyV neutralizing activity. Relevant consequences may derive from future clinical studies induced by these findings; indeed the study of the serum anti-JCPyV neutralizing activity could allow not only a better risk stratification of the patients during natalizumab treatment, but also a better understanding of the pathophysiological mechanisms leading to PML, highlighting the contribution of peripheral versus central nervous system JCPyV reactivation. Noteworthy, the availability of GRE1 could allow the design of novel immunoprophylactic strategies during the immunomodulatory treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. cDNA cloning, characterization and expression analysis of DTX2, a human WWE and RING-finger gene, in human embryos.

    Science.gov (United States)

    Yi, Zhengfang; Yi, Tingfang; Wu, Zirong

    2006-06-01

    The WWE domain is a conserved globular domain in several proteins and predicted to mediate specificprotein-protein interactions in ubiquitin and ADP ribose conjugation systems. The RING domain is a conserved and specialized zinc-finger motif with 40-60 residues binding to two zinc atoms, which is also probably involved in mediating protein-protein interactions. Here, from human fetal heart cDNA library, we identified DTX2, a human WWE & RING-finger gene, with high similarity with its homologues. Evaluation of full-length cDNA obtained by RACE indicated it encodes a protein composed of two WWE domains and a RING-finger region. The DTX2 gene located in human chromosome 7q11.23 spanning approximately 44.3 kb on the genome and the deduced protein is 622 amino acids. Northern analysis revealed DTX2 was expressed in the 18-week, 22.5-week human embryo hearts and adult hearts, especially with high levels in the 18-week and adult hearts. Taken together, these results indicate that DTX2 is a gene encoding a WWE-RING-finger protein and involved in regulating heart development and heart functions.

  6. Human papillomaviruses associated with epidermodysplasia verruciformis. II. Molecular cloning and biochemical characterization of human papillomavirus 3a, 8, 10, and 12 genomes.

    OpenAIRE

    Kremsdorf, D; Jablonska, S.; Favre, M.; Orth, G

    1983-01-01

    The DNAs of four human papillomaviruses (HPVs) that were found in the benign lesions of three patients suffering from epidermodysplasia verruciformis have been characterized. The flat wart-like lesions and the macular lesions of patient 1 contained two viruses, HPV-3a and HPV-8, respectively, whose genomes had previously been only partially characterized. The flat wart-like lesions of patient 2 and the macular lesions of patient 3 each contained a virus previously considered as belonging to t...

  7. Molecular cloning and chromosomal localization of the human cyclin C (CCNC) and cyclin E (CCNE) genes: Deletion of the CCNC gene in human tumors

    Energy Technology Data Exchange (ETDEWEB)

    Li, Haimin; Lahti, J.M.; Kidd, V.J. [St. Jude Children`s Research Hospital, Memphis, TN (United States)] [and others

    1996-03-01

    The human G1-phase cyclins are important regulators of cell cycle progression that interact with various cyclin-dependent kinases and facilitate entry into S-phase. We have confirmed the localization of the human cyclin C (CCNC) gene to chromosome 6q21 and of human cyclin E (CCNE to 19q12). The CCNC gene structure was also determined, and we have shown that it is deleted in a subset of acute lymphoblastic leukemias, including a patient sample containing a t(2;6)(p21;q15), with no apparent cytogenetic deletion. Single-strand conformational polymorphism analysis of the remaining CCNC allele from patients with a deletion of one allele established that there were no further mutations within the exons or the flanking intronic sequences. These results suggest either that haploinsufficiency of the cyclin C protein is sufficient to promote tumorigenesis or that the important tumor suppressor gene is linked to the CCNC locus. 48 refs., 4 figs., 1 tab.

  8. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Directory of Open Access Journals (Sweden)

    Kathy N Lam

    Full Text Available High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  9. Evaluation of a pooled strategy for high-throughput sequencing of cosmid clones from metagenomic libraries.

    Science.gov (United States)

    Lam, Kathy N; Hall, Michael W; Engel, Katja; Vey, Gregory; Cheng, Jiujun; Neufeld, Josh D; Charles, Trevor C

    2014-01-01

    High-throughput sequencing methods have been instrumental in the growing field of metagenomics, with technological improvements enabling greater throughput at decreased costs. Nonetheless, the economy of high-throughput sequencing cannot be fully leveraged in the subdiscipline of functional metagenomics. In this area of research, environmental DNA is typically cloned to generate large-insert libraries from which individual clones are isolated, based on specific activities of interest. Sequence data are required for complete characterization of such clones, but the sequencing of a large set of clones requires individual barcode-based sample preparation; this can become costly, as the cost of clone barcoding scales linearly with the number of clones processed, and thus sequencing a large number of metagenomic clones often remains cost-prohibitive. We investigated a hybrid Sanger/Illumina pooled sequencing strategy that omits barcoding altogether, and we evaluated this strategy by comparing the pooled sequencing results to reference sequence data obtained from traditional barcode-based sequencing of the same set of clones. Using identity and coverage metrics in our evaluation, we show that pooled sequencing can generate high-quality sequence data, without producing problematic chimeras. Though caveats of a pooled strategy exist and further optimization of the method is required to improve recovery of complete clone sequences and to avoid circumstances that generate unrecoverable clone sequences, our results demonstrate that pooled sequencing represents an effective and low-cost alternative for sequencing large sets of metagenomic clones.

  10. 28 CFR 42.510 - Discrimination prohibited.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Discrimination prohibited. 42.510 Section...-Implementation of Section 504 of the Rehabilitation Act of 1973 Employment § 42.510 Discrimination prohibited. (a) General. (1) No qualified handicapped person shall on the basis of handicap be subjected to discrimination...

  11. 5 CFR 900.704 - Discrimination prohibited.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 2 2010-01-01 2010-01-01 false Discrimination prohibited. 900.704... Federally Assisted Programs of the Office of Personnel Management § 900.704 Discrimination prohibited. (a..., be denied the benefits of, or otherwise be subjected to discrimination under any program or activity...

  12. 38 CFR 18.411 - Discrimination prohibited.

    Science.gov (United States)

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Discrimination prohibited... Practices § 18.411 Discrimination prohibited. (a) General. (1) No qualified handicapped person shall, on the basis of handicap, be subjected to discrimination in employment under any program or activity to which...

  13. 22 CFR 217.11 - Discrimination prohibited.

    Science.gov (United States)

    2010-04-01

    ... 22 Foreign Relations 1 2010-04-01 2010-04-01 false Discrimination prohibited. 217.11 Section 217... Discrimination prohibited. (a) General. (1) No qualified handicapped person shall, on the basis of handicap, be subjected to discrimination in employment under any program or activity to which this part applies. (2) A...

  14. 28 CFR 42.203 - Discrimination prohibited.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Discrimination prohibited. 42.203 Section...) of the Justice System Improvement Act of 1979 § 42.203 Discrimination prohibited. (a) No person in... participation in, be denied the benefits of, be subjected to discrimination under, or denied employment in...

  15. 45 CFR 605.11 - Discrimination prohibited.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Discrimination prohibited. 605.11 Section 605.11... Employment Practices § 605.11 Discrimination prohibited. (a) General. (1) No qualified handicapped person shall, on the basis of handicap, be subjected to discrimination in employment under any program or...

  16. 45 CFR 605.21 - Discrimination prohibited.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Discrimination prohibited. 605.21 Section 605.21... Accessibility § 605.21 Discrimination prohibited. No qualified handicapped person shall, because a recipient's... from participation in, or otherwise be subjected to discrimination under any program or activity to...

  17. 5 CFR 900.404 - Discrimination prohibited.

    Science.gov (United States)

    2010-01-01

    ... 5 Administrative Personnel 2 2010-01-01 2010-01-01 false Discrimination prohibited. 900.404... § 900.404 Discrimination prohibited. (a) General. A person in the United States shall not, on the ground... be otherwise subjected to discrimination under, a program to which this subpart applies. (b) Specific...

  18. 43 CFR 17.203 - Discrimination prohibited.

    Science.gov (United States)

    2010-10-01

    ... 43 Public Lands: Interior 1 2010-10-01 2010-10-01 false Discrimination prohibited. 17.203 Section... Discrimination prohibited. (a) General. No qualified handicapped person shall, on the basis of handicap, be excluded from participation in, be denied the benefits of, or otherwise be subjected to discrimination...

  19. 38 CFR 18.404 - Discrimination prohibited.

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    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Discrimination prohibited... Provisions § 18.404 Discrimination prohibited. (a) General. No qualified handicapped person shall, on the... subjected to discrimination under any program or activity which receives Federal financial assistance. (b...

  20. 38 CFR 18.421 - Discrimination prohibited.

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    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Discrimination prohibited... Accessibility § 18.421 Discrimination prohibited. No qualified handicapped person shall, because a recipient's... from participation in, or otherwise be subjected to discrimination under any program or activity to...