Somatic Cell Genetics Research Articles

Induction and characterization of induced tetraploid, mixoploid and control diploid teak seedlings (Tectona grandis L.f.) from shoot cultures treated with oryzalin*

Teak, known for its high-quality timber and economic value, holds significant potential as the major component of an agroforestry system to combat climate change and support sustainable development goals. The challenge of slow teak growth can be addressed by introducing fast-growing teak varieties amenable to vegetative propagation. Furthermore, the improvement of teak through somatic cell genetics, such as inducing polyploidy, can result in more vigorous, faster-growing, and drought-tolerant trees. While a report exists on a tetraploid teak accession with enhanced less water tolerance, information on the induction and characterization of tetraploid teak is lacking. In this study, we report the induction of polyploid teak by immersing 1 – 1.5 cm nodal stem segments and shoot tips obtained from teak shoot cultures in a modified MS liquid medium containing 5 − 30 µM oryzalin for one week, followed by subculture for 6 passages. The resulting shoots were acclimatized and grown in the nursery for 10 months. Based on flow cytometry for ploidy analysis of pre-selected plants having morphologies typical of polyploid followed by chromosome counting, we identified two tetraploid plants from one accession and 18 mixoploid teak plants from four accessions. Chromosome counting analysis confirmed that a diploid plant had a chromosome number of 2n = 2x = 36, tetraploid plant had a chromosome number of 2n = 4x = 72, and mixoploid plant exhibited both chromosome number of 2n = 2x = 36 and 2n = 4x = 72 in root tip cells. Morphological characterization revealed that tetraploid and mixoploid teak plants displayed typical polyploid characteristics, such as larger, thicker, and greener leaves, increased root diameter, increased cell size in root tip region, and larger stomata size but with reduced density. The development of tetraploid teak holds the potential to provide faster-growing and more drought-tolerant varieties, facilitating breeding efforts, including the development of triploid varieties.

Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp.

Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

Traptamer screening: a new functional genomics approach to study virus entry and other cellular processes.

Historically, the genetic analysis of mammalian cells entailed the isolation of randomly arising mutant cell lines with altered properties, followed by laborious genetic mapping experiments to identify the mutant gene responsible for the phenotype. In recent years, somatic cell genetics has been revolutionized by functional genomics screens, in which expression of every protein-coding gene is systematically perturbed, and the phenotype of the perturbed cells is determined. We outline here a novel functional genomics screening strategy that differs fundamentally from commonly used approaches. In this strategy, we express libraries of artificial transmembrane proteins named traptamers and select rare cells with the desired phenotype because, by chance, a traptamer specifically perturbs the expression or activity of a target protein. Active traptamers are then recovered from the selected cells and can be used as tools to dissect the biological process under study. We also briefly describe how we have used this new strategy to provide insights into the complex process by which human papillomaviruses enter cells.

NS5A Promotes Constitutive Degradation of IP3R3 to Counteract Apoptosis Induced by Hepatitis C Virus

FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3's degron in the absence of inositol 1,4,5-trisphosphate(IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85β, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppressionof HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.

Cell population genetics: emergence, main results and concepts (to the 50th anniversary of the foundation)

The article briefly reviews the emergence and development in Ukraine of a new scientific field — cell population genetics, which formed the theoretical basis of modern cell technologies. These include, in particular, plant biotechnologies for the improvement, preservation, and accelerated reproduction of unique genotypes in vitro; development of new genotypes (organisms) by the methods of cell and genetic engineering and cellular selection; obtaining biologically active compounds, including recombinant, from biomass of cultured cells and tissues for the needs of medicine, cosmetics and food industry; as well as the methods of cell therapy, including technologies based on the use of stem cells, etc. Cultured cells are widely used as model objects and biological systems for studying the most relevant problems of modern biology: the features of the course, signaling pathways, and mechanisms of cell proliferation, including carcinogenesis and tumor proliferation; dedifferentiation of cells, including their reversion to a pluripotent state; totipotency, pluripotency and omnipotency; regeneration of tissues, separate organs, and whole organisms, etc. The article reviews scientific prerequisites for the development of the new scientific field and presents the main concepts of cell population genetics, which have been mainly developed in the Department of cell population genetics of the Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine. It also presents an analysis of the features of development of the newest research areas of genetics of somatic cells of intact plants and cells in vitro, cell population genetics, genetic foundations of cell selection, cell biology, and biotechnology during the second half of the past and the beginning of this century. Keywords: history of science, cell population genetics, plant tissue and cell culture, cell selection, plant biotechnology.

Molecular Analysis Of Mitochondrial Dynamics

The dynamics properties of mitochondria control their function. I will update our efforts to understand the molecular mechanisms of mitochondrial dynamics and their physiological functions. I will also discuss our use of somatic cell genetics to identify genes involved in mitochondrial function. This includes our attempt to understand why cancer cells are dependent on glucose metabolism. In such a screen, we identified the xCT antiporter, which plays an antioxidant role by exporting glutamate for cystine. Disruption of the xCT antiporter greatly improves cell viability after glucose withdrawal, because conservation of glutamate enables cells to maintain mitochondrial respiration. In some breast cancer cells, xCT antiporter expression is upregulated through the antioxidant transcription factor Nrf2 and contributes to their requirement for glucose as a carbon source.

The historical role of species from the Solanaceae plant family in genetic research.

Key message This article evaluates the main contributions of tomato, tobacco, petunia, potato, pepper and eggplant to classical and molecular plant genetics and genomics since the beginning of the twentieth century. Species from the Solanaceae family form integral parts of human civilizations as food sources and drugs since thousands of years, and, more recently, as ornamentals. Some Solanaceous species were subjects of classical and molecular genetic research over the last 100 years. The tomato was one of the principal models in twentieth century classical genetics and a pacemaker of genome analysis in plants including molecular linkage maps, positional cloning of disease resistance genes and quantitative trait loci (QTL). Besides that, tomato is the model for the genetics of fruit development and composition. Tobacco was the major model used to establish the principals and methods of plant somatic cell genetics including in vitro propagation of cells and tissues, totipotency of somatic cells, doubled haploid production and genetic transformation. Petunia was a model for elucidating the biochemical and genetic basis of flower color and development. The cultivated potato is the economically most important Solanaceous plant and ranks third after wheat and rice as one of the world’s great food crops. Potato is the model for studying the genetic basis of tuber development. Molecular genetics and genomics of potato, in particular association genetics, made valuable contributions to the genetic dissection of complex agronomic traits and the development of diagnostic markers for breeding applications. Pepper and eggplant are horticultural crops of worldwide relevance. Genetic and genomic research in pepper and eggplant mostly followed the tomato model. Comparative genome analysis of tomato, potato, pepper and eggplant contributed to the understanding of plant genome evolution.

Abstract IA28: Epigenetic heterogeneity in breast cancer

Abstract Heterogeneity of cancer cells between and within tumors is a major obstacle toward understanding and treatment of cancers. Variability of epigenetic states is one of the major contributor to both intertumor and intratumor heterogeneity. In breast cancer luminal and basal-like breast tumors have very different molecular and clinical profiles. However, even in individual tumors there is variability among cells for these features. The determinants of luminal cell phenotypes are fairly well understood and major transcriptional regulators have been identified. In contrast, transcriptional determinants of basal-like breast tumors are still poorly defined and high variability is observed among individual tumors. We have investigated molecular regulators of luminal and basal-like phenotypes by applying somatic cell genetics and cellular reprogramming approaches combined with comprehensive molecular profiling. We found that the basal-like state is predominantly dominant and it is defined mainly by epigenetic mechanisms. We have also identified candidate epigenetic and transcriptional regulators of basal-like cellular states. Modulation of epigenetic regulators may decrease intratumor heterogeneity and in combination with other agents could improve the efficacy of cancer therapies. Citation Format: Kornelia Polyak. Epigenetic heterogeneity in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA28.

Sequence determinants of improved CRISPR sgRNA design.

The CRISPR/Cas9 system has revolutionized mammalian somatic cell genetics. Genome-wide functional screens using CRISPR/Cas9-mediated knockout or dCas9 fusion-mediated inhibition/activation (CRISPRi/a) are powerful techniques for discovering phenotype-associated gene function. We systematically assessed the DNA sequence features that contribute to single guide RNA (sgRNA) efficiency in CRISPR-based screens. Leveraging the information from multiple designs, we derived a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 knockout experiments. Our model confirmed known features and suggested new features including a preference for cytosine at the cleavage site. The model was experimentally validated for sgRNA-mediated mutation rate and protein knockout efficiency. Tested on independent data sets, the model achieved significant results in both positive and negative selection conditions and outperformed existing models. We also found that the sequence preference for CRISPRi/a is substantially different from that for CRISPR/Cas9 knockout and propose a new model for predicting sgRNA efficiency in CRISPRi/a experiments. These results facilitate the genome-wide design of improved sgRNA for both knockout and CRISPRi/a studies.

A Mathematician's Odyssey.

In this overview of my research, I have aimed to give the background as to how I came to be involved in my various areas of interest, with an emphasis on the early phases of my career, which largely determined my future directions. I had the enormous good fortune to have worked under two of the most outstanding scientists of the twentieth century, R.A. Fisher and Joshua Lederberg. From mathematics and statistics, I went to population genetics and the early use of computers for modeling and simulation. Molecular biology took me into the laboratory and eventually to somatic cell genetics and human gene mapping. One chance encounter led me into the HLA field and another led me into research on cancer, especially colorectal cancer. On the way, I became a champion of the Human Genome Project and of the need for scientists to help promote the public understanding of science.

Twenty years of RNA: reflections from the RNP world.

Twenty years of RNA: reflections from the RNP world.

A pathway from chromosome transfer to engineering resulting in human and mouse artificial chromosomes for a variety of applications to bio-medical challenges.

Microcell-mediated chromosome transfer (MMCT) is a technique to transfer a chromosome from defined donor cells into recipient cells and to manipulate chromosomes as gene delivery vectors and open a new avenue in somatic cell genetics. However, it is difficult to uncover the function of a single specific gene via the transfer of an entire chromosome or fragment, because each chromosome or fragment contains a set of numerous genes. Thus, alternative tools are human artificial chromosome (HAC) and mouse artificial chromosome (MAC) vectors, which can carry a gene or genes of interest. HACs/MACs have been generated mainly by either a “top-down approach” (engineered creation) or a “bottom-up approach” (de novo creation). HACs/MACs with one or more acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native chromosomes. The MMCT technique is also applied for manipulating HACs and MACs in donor cells and delivering them to recipient cells. This review describes the lessons learned and prospects identified from studies on the construction of HACs and MACs, and their ability to drive exogenous gene expression in cultured cells and transgenic animals via MMCT. New avenues for a variety of applications to bio-medical challenges are also proposed.

Forward genetic screening for regulators involved in cholesterol synthesis using validation-based insertional mutagenesis.

Somatic cell genetics is a powerful approach for unraveling the regulatory mechanism of cholesterol metabolism. However, it is difficult to identify the mutant gene(s) due to cells are usually mutagenized chemically or physically. To identify important genes controlling cholesterol biosynthesis, an unbiased forward genetics approach named validation-based insertional mutagenesis (VBIM) system was used to isolate and characterize the 25-hydroxycholesterol (25-HC)-resistant and SR-12813-resisitant mutants. Here we report that five mutant cell lines were isolated. Among which, four sterol-resistant mutants either contain a truncated NH2-terminal domain of sterol regulatory element-binding protein (SREBP)-2 terminating at amino acids (aa) 400, or harbor an overexpressed SREBP cleavage-activating protein (SCAP). Besides, one SR-12813 resistant mutant was identified to contain a truncated COOH-terminal catalytic domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). This study demonstrates that the VBIM system can be a powerful tool to screen novel regulatory genes in cholesterol biosynthesis.

Serendipity or prepared mind? Recollections of the KOP translocation (1967) and of one form of Perrault syndrome.

The human X/autosome translocation, designated KOP, was discovered by Dr. Philip D. Pallister in Montana in 1967 in a young man with apparent Klinefelter syndrome. Collaboratively it was possible to elucidate the genetic nature of his unprecedented chromosome rearrangement and its developmental effects in mother and son. In retrospect, these clinical and genetic studies at the height of the somatic cell genetics era (Ruddle, Siniscalco, etc.) presented human genetics with a highly productive opportunity to begin gene mapping of autosomes and the X chromosome. The late Victor McKusick considered the discovery of the KOP translocation, as he determined personally in Montana, one of the major transforming events in human genetics. The Perrault syndrome evaluated in two families in Montana and one in Sicily for familial deafness, primary amenorrhea and neurologic impairment (progressive in some), turned out to be heterogeneous. In the "hands" of Dr. M.-C. King of Seattle four forms of Perrault syndrome have been identified. The autosomal recessive mutation present in the P family studied with Dr. Pallister in Helena, turned out to affect the mitochondrial histidyl tRNA synthetase gene present in prokaryotes, annelids, fungi and mammals, hence, must already have been present in LUCA some 3.8 billion years ago.

Phenotypic Changes in Growth-Arrested T Cell Hybrids: A Possible Avenue to Produce Functional T Cell Hybridoma

Attempts to immortalize effector T cells by fusion of normal T cells with T cell lymphomas have been extensively made after the successful establishment of the B cell hybridoma that made it possible to infinitely produce monoclonal antibodies (1, 2). However, T cell hybridomas, which were mostly produced by using the T cell lymphoma line, BW5147, have contributed to T cell biology with limited success, because immortalizing effector T cells by cell fusion dose not necessarily result in immortalizing the bona fide effector functions of parental T cells. Meanwhile, T cells have been becoming recognized as consisting of more diverse populations than previously anticipated (3–6). Furthermore, a new class of lymphocytes termed innate lymphoid cells has been identified; they lack rearranged antigen receptors and are involved not only in immunity but also in inflammation and in tissue remodeling and repair (6–9). Thus, in order to gain a better understanding of the functions of various lymphocytes and their roles in tissue microenvironment, it is desirable to produce functional hybridomas that preserve the effector functions of the parental lymphocytes faithfully. We briefly review phenotypic changes in T cell hybrids from the point of view of somatic cell genetics and describe the concepts and principles behind devising an improved method for immortalizing various lymphocyte functions.

The Na+/H+ exchangers of intracellular compartments. Proton leaks or loading mechanisms ?

Event Abstract Back to Event The Na+/H+ exchangers of intracellular compartments. Proton leaks or loading mechanisms ? Nina Milosavljevic1, MIchael Monet2, Isabelle Lena2, Frederic Brau3, Sandra Lacas-Gervais4, Sylvain Feliciangeli3, Mallorie Poet2 and Laurent Counillon2* 1 University of Manchester, Faculty of Life Sciences, United Kingdom 2 University of Nice-Sophia Antipolis, Biology, France 3 IPMC, UMR 7275, France 4 CCMA, France Background and aim. The Na+/H+ exchangers NHE 7, 8 and 9 are expressed in intracellular vesicles. NHE7 and NHE6 genes are situated on the X chromosome (Xp11.3 and Xq26.3, respectively) and NHE9 is on chromosome 3 (3q24). Mutations in the genes encoding these exchangers have been linked to autism-spectrum neurological disorders and neurodegeneration. In addition, the overexpression of NHE7 in human breast carcinoma cells enhances cell-cell adhesion, anchorage-independent tumor growth, invasion and tumor formation in vivo. These transporters are mostly believed to exchange cytosolic potassium for protons and to alkalinize vesicles, an important function for the maintenance of steady state pH in intracellular compartments. However, their intracellular localization prevented direct functional measurements. Methods. Using somatic cell genetics, we selected a cell line that expresses wild-type NHE7 at the plasma membrane. This enabled for the first time the detailed measurement of the exchanger’s parameters and constants using fast kinetics of ion transport. Conclusions / Discussion. These measurements show unexpected functions for NHE7 and provide new clues for the biological roles and pathological implications of these intracellular NHEs. Keywords: modelling, Membranes, Proton transporters, Vesicular Transport Proteins, human diseases Conference: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer, Garching, Germany, 10 Oct - 12 Oct, 2013. Presentation Type: Abstract Topic: 1. Biophysics of pH Citation: Milosavljevic N, Monet M, Lena I, Brau F, Lacas-Gervais S, Feliciangeli S, Poet M and Counillon L (2014). The Na+/H+ exchangers of intracellular compartments. Proton leaks or loading mechanisms ?. Front. Pharmacol. Conference Abstract: 4th Annual Meeting of the International Society of Proton Dynamics in Cancer. doi: 10.3389/conf.fphar.2014.61.00030 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 23 Jan 2014; Published Online: 07 Feb 2014. * Correspondence: Prof. Laurent Counillon, University of Nice-Sophia Antipolis, Biology, Nice, 06108, France, Laurent.Counillon@unice.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nina Milosavljevic MIchael Monet Isabelle Lena Frederic Brau Sandra Lacas-Gervais Sylvain Feliciangeli Mallorie Poet Laurent Counillon Google Nina Milosavljevic MIchael Monet Isabelle Lena Frederic Brau Sandra Lacas-Gervais Sylvain Feliciangeli Mallorie Poet Laurent Counillon Google Scholar Nina Milosavljevic MIchael Monet Isabelle Lena Frederic Brau Sandra Lacas-Gervais Sylvain Feliciangeli Mallorie Poet Laurent Counillon PubMed Nina Milosavljevic MIchael Monet Isabelle Lena Frederic Brau Sandra Lacas-Gervais Sylvain Feliciangeli Mallorie Poet Laurent Counillon Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Len Herzenberg (1931–2013)

Leonard Arthur Herzenberg was born the 5th November 1931 in New York, USA. He studied Biology and Chemistry at Brooklyn College. Len did his PhD in biochemistry and immunology at the CalTech (Pasadena, CA, USA). When he finished his PhD in 1955 he moved to France for a Postdoc in the Institute Pasteur, where he worked with Jacques Monod. In 1957, he joined Harry Eagle at the National Institutes of Health (NIH), adding pyruvate to the recipe of “Eagle's medium”, and working on somatic cell genetics of cell lines. In 1959, Joshua Lederberg recruited Len to the Department of Genetics of the Stanford University (CA, USA). At that time, when somatic cell genetics were introduced into immunology, Len worked on H-2 and IgH genetics, in particular on IgH allotypes. The lack of methods to isolate rare genetic variants from cell lines was a limitation for his studies. Looking for technologies to solve this bottleneck, Len discovered that a group of engineers headed by Mack Fulwyler at Los Alamos National Laboratories (New Mexico, USA) had developed a particle sorter by combining a Coulter volume sensor with an ink jet printer device. Len asked them to add a detector for fluorescence of the particles, but they rather gave him the construction plans. Len collected the 14 000 $ required to build a prototype at the Stanford instrumentation laboratory. This prototype was presented in 1969 1, improved and in 1972, the instrument was baptized “Fluorescence-activated Cell Sorter”, also known as FACS. Soon, it turned out to be a key instrument for the analysis of the immune system. For the first time ever, FACS allowed the quantitation of expression of a particular protein in individual cells, stained with fluorescent, specific antibodies, and the isolation of individual cells according to their protein expression. Soon several proteins could be detected simultaneously, using antibodies with different fluorescent dyes. It was a breakthrough for cytometry and immunology. And although todays “flow-in-air” FACS machines incorporate all kind of devices not available in the 70's, they still operate according to the original principle. Today, the speed of sorting has increased from 2 000 cells per second to 40 000 cells per second, and the number of fluorescent parameters from 1 to 20. There is hardly no modern scientific concept in immunology that has not benefited from cytometric immunofluorescence and cell sorting. I personally, as a young scientist, was completely dependent on FACS, to isolate somatic variants of immunoglobulin expression for a molecular analysis of somatic hypermutation and antibody class switching 2. And the MACS technology was essentially developed to complement FACS too 3. The FACS technology continues to challenge engineers and immunologists alike and this is Len's heritage 4. At the beginning, two points were essential for the success of the FACS. First, Len succeeded in convincing Becton-Dickinson (BD) to commercialize the FACS technology, though initially, neither he nor BD believed that there was really a huge market for such machines 5. Second, the advent of monoclonal antibodies in 1975 was catapulting immunofluorescence into a new era. Len immediately recognized that, and in 1976 spent a sabbatical with Cesar Milstein at the MRC in Cambridge, UK. He returned then to Stanford with the new monoclonal antibody technology. The triade of immunofluorescence, cytometry and cell sorting became a unique and precious tool that we are still using today. Len himself, together with Lee, his wife of 60 years, and a host of talented young scientists, used the FACS technology from the start, to dissect the immune system. They visualized allelic exclusion, B cells as plasma cell precursors, discovered B1 cells, T-cell imbalances in diseases like HIV and isolated genetic variants of hybridoma cell lines. In short, they contributed essentially to our current understanding of the cellularity of the immune system 5. For his seminal contributions to immunology, Len was awarded with the Novartis Immunology prize and the Kyoto prize. Len's activities were not restricted to science, though. He was severely concerned about and reacting to McCarthyism, eugenics and HIV stigmas, activities he considered to be the responsibility of a scientist 6. Len Herzenberg died on the 27th of October 2013. Immunology has lost a great personality and scientist, an inspiring mentor and a dear friend to many of us!

A novel haploid genetics approach to dissect immune pathways (P3235)

Abstract Unbiased loss-of-function genetic screens in model organisms have been major driving forces for modern biological research. However, many biological functions of humans such as adaptive immunity are not found in model organisms amenable to classic genetic screens. Despite tremendous efforts, human somatic cell genetics remains unattainable due to the inability to efficiently induce homozygous mutations in diploid human cells. Our group developed a novel haploid genetics approach to dissect immune pathways in human cells. We take advantage of human myeloid cells lines (e.g. KBM7) that exhibit a haploid karyotype. Retroviral insertional mutagenesis of the haploid cells generates a library of mutant cells with individual genes completely disrupted. First, we validated the haploid mutant library by screening for mutants resistant to the cytotoxicity of diphtheria toxin. After selection, the retroviral insertion sites of the resistant clones were mapped by deep sequencing. We identified HBEGF, the known cell surface receptor for diphtheria toxin. We also isolated multiple endocytic membrane proteins, which likely mediate the internalization of diphtheria toxin. Next, we demonstrated that the haploid mutant library is also suitable for studying complex immune pathways such as the elimination of tumor cells by cytotoxic T lymphocytes. This haploid genetics approach provides a novel and powerful platform for dissecting immune pathways on a genome-wide scale.

Drawing the Map of Life inside the Human Genome Project (review)

Reviewed by: Drawing the Map of Life inside the Human Genome Project Nathaniel Comfort (bio) Drawing the Map of Life inside the Human Genome Project. By Victor McElheny. New York: Basic Books, 2010. Pp. 362. $28. The history of recent science is the second draft of history. That gives we who write it a special relationship to science reporters. We don’t have the luxury of dismissing them as unserious; often, their narratives are what those of us who work the leading edge of history must reinforce, revise, or refute. Since the 1970s Victor McElheny has chronicled the maturation of molecular biology from the thrilling vanguard of laboratory science into an economic powerhouse and the core discipline of biomedicine. His 2003 life of James Watson is the first biography of the celebrated co-solver of the structure of DNA. In his latest book McElheny draws on firsthand experience, news reports and press releases, interviews, the scientific literature, and some scholarly books in recounting the origins of the Human Genome Project and bringing its story up-to-date. He chooses to emphasize the genome as technology, an approach that, without some sophistication, easily leads to a triumphalist narrative—a seduction that McElheny makes no visible effort to resist. He begins, appropriately enough, in the 1970s, with the development of restriction enzymes, recombinant DNA, model organisms, and, of course, DNA sequencing. But then, inexplicably, he leaps to the 1993 identification of the Huntington’s disease gene—sometimes called the “crown jewel” of gene-hunting—before backtracking to deliver a quick briefing on the development of the biotechnology industry. With such balletic celebration McElheny flies past some critical techniques, such as human–mouse cell hybridization, invented by Mary Weiss and Howard Green and developed by Frank Ruddle and others, into the first effective means of mapping auto-somal human genes. Somatic cell genetics is not part of the canonical genome narrative. In fact, the entire narrative of clinical gene-mapping and the Human Genome Organization, so crucial to shaping the early genome project, is barely mentioned in this or other genome histories. McElheny is more interested in hitting the highlights than in conveying the richness of the history as it appeared while it was unfolding. The significant debate over whether to undertake a “big science” genome project is gathered under the rubric of “getting started.” If this book represents the historiographical trend, the disciplinary soul-searching of the late 1980s, so trenchantly covered by Robert Cook-Deegan in 1994 in The Gene Wars, is coming to be crammed into the prehistory of the genome. Perhaps it is becoming difficult, given the transformative effect of genomics on biology, to see that early dissent as much more than shortsightedness. Here, the project’s “crisis,” rather, comes in the late 1990s when Craig Venter turned the project on its head. Short-circuiting previous debates over which parts of the genome to sequence first and who had the most [End Page 240] expertise on which chromosome, Venter formed a private company and proposed to sequence the entire genome in one go. Or rather, a series of goes, cross-checked against one another to provide redundancy and therefore increased confidence. His approach telescoped the genome project from a ten-year slog into a mad three-year dash to a first draft of the genome in 2000. The second half of the book covers the fast-moving period since the turn of the century. This is the best part, since here the historiographical trail fades and one can easily feel lost in the jungle. Helpfully, McElheny roughs out many important moments of this dynamic, transitional period: the drastic downsizing of our estimates of the number of human genes; the surprising discovery of new and potent, though short-lived, forms of RNA; and the realization that the genome was far from a static “blueprint” or “parts manual.” It now seems a richly dynamic system, constantly rearranging itself and responding to the environment. The genome truly is, as Barbara McClintock said in 1980, a “sensitive organ of the cell.” This draft of history is quite rough, however. In the last, most valuable third, the narrative largely dissolves and the book becomes...

Characterization of macrophage mutants established by their resistance to LPS and cycloheximide-induced apopotic cell death

Macrophages are activated by bacterial lipopolysaccharide (LPS) to produce inflammatory cytokines such as TNF-α or reactive oxygen species such as nitric oxide or superoxide anion. However, in the presence of an inhibitor of protein synthesis, cyclohex-imide (CHX), at 10 μg/mL, LPS at 100 ng/mL induced macrophage apoptosis rapidly without producing phenotypes of activated macrophages. In order to understand the mechanism underlying LPS-induced cytotoxicity toward macrophages, we isolated mutant cells from a macrophage-like cell line, J774.1, as clones resistant against the cytotoxic effects of LPS + CHX by using a somatic cell genetics protocol. All of the mutant clones, designated as LCR mutants, showed resistance to the cell death induced by LPS + CHX as well as to that induced by higher doses of LPS alone, as did the LPS1916 mutant cell line, which had been previously established by its resistance to 100 μg/mL LPS. Characterization of the activated macrophage phenotypes revealed that these mutants showed reduced production of TNF-α and nitric oxide in response to LPS. Further analysis showed a much reduced amount of [125I]LPS-binding and lower CD14 expression on the cell surface, in spite of an adequate intracellular expression of CD14 molecules. Besides, the molecular weight of CD14 on these mutants was around 40-48 kDa, smaller than that of the wild-type JA-4 cells (around 50-55 kDa), suggesting impaired CD14 maturation in these mutants. However, expression of Toll-like receptor 4 (TLR4) and Myd 88 on the cell surface was not different between the wild type and the mutant cells. These results suggest that LCR mutants have common phenotypes of mal-expression of CD14 molecules on the macrophage cell surface, leading to not only reduced responses to LPS-mediated macrophage activation but acquisition of resistance to LPS-induced apoptotic cell death in the presence of CHX.