DOCSLIB.ORG

Efficient Recovery and Sequencing of Mutant Genes from Mammalian Chromosomal DNA (Mutagenesis/Retroviral Vector/Base Substitution/Deletion) CHARLES R

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Efficient Recovery and Sequencing of Mutant Genes from Mammalian Chromosomal DNA (Mutagenesis/Retroviral Vector/Base Substitution/Deletion) CHARLES R Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3356-3360, May 1986 Genetics Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA (mutagenesis/retroviral vector/base substitution/deletion) CHARLES R. ASHMAN, PUDUR JAGADEESWARAN, AND RICHARD L. DAVIDSON Center for Genetics, University of Illinois College of Medicine at Chicago, 808 South Wood Street, Chicago, IL 60612 Communicated by Theodore T. Puck, December 26, 1985 ABSTRACT A retroviral shuttle vector was constructed by was mutagen-induced. In addition, the selectable gene is introducing the Escherichia coli xanthine (guanine) phospho- integrated into chromosomal DNA in the mammalian cells ribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector rather than being maintained in an autonomously replicating [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) CeU molecule. 37, 1053-1062]. This vector was packaged into infectious virus The selectable gene used in this study is the E. coli gpt which then was used to infect a hypoxanthine (guanine) gene, which codes for xanthine (guanine) phosphoribosyl- phosphoribosyltransferase-deficient mouse cell line. Cell lines transferase (XPRTase). This gene was chosen because there that expressed the gpt gene were isolated, and it was found that are selective systems for and against XPRTase activity in these cells contained a single integrated copy of the vector in a both bacteria and mammalian cells. The gpt gene was proviral form. Treatment of these cell lines with either ethyl inserted into the retroviral shuttle vector pZip-NeoSV(X)1 methanesulfonate or BrdUrd produced a >10-fold increase in (8), which can be packaged into a virus that integrates into the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact chromosomal DNA of infected cells. Also, the vector se- gpt genes have been recovered from a number of Sgur cell lines quences can be efficiently recovered from infected cells and after COS cell fusion and introduced into E. coli as part of a introduced into E. coli as plasmids. In this paper we describe plasmid. The complete DNA sequences of three mutant genes a system that uses this retroviral shuttle vector to rescue have been determined. Two of the mutant genes have a single mutant gpt genes from mammalian cells, and we have base substitution, whereas the third has a 34-base-pair dele- sequenced three mutant genes recovered after mutagenesis. tion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical MATERIALS AND METHODS mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is Cell Culture, DNA Transfection, and Virus Infection. Line that the mutations are induced in genes integrated into mam- A9 is a hypoxanthine (guanine) phosphoribosyltransferase malian chromosomes rather than in genes existing as part of (HPRTase)-deficient derivative of mouse L cells (9). COS-1 autonomously replicating plasmids. cells (10) were provided by K. Subramanian and psi-2 cells (11) by R. Mulligan. The basic cell culture medium was "Shuttle vectors" capable of replication in both animal cells Dulbecco's modified Eagle's medium supplemented with and bacteria have been used to study mutagenesis in mam- 10% fetal bovine serum; psi-2 cells were grown in medium malian cells (1-7). The basic strategy has been to introduce supplemented with 10% calf serum. HAT medium (12) is a marker bacterial gene into mammalian cells as part of a supplemented with 0.1 mM hypoxanthine, 0.4 mM amino- shuttle vector, recover populations of shuttle-vector mole- pterin, and 16 AM thymidine. Selections for G418 (13), cules from the cells, transform Escherichia coli, and score 6-thioguanine (Sgu), and ouabain resistance were carried out mutants in the marker gene in the E. coli transformants. The in medium supplemented with 1 mM G418, 36 pzM Sgu, or 1 major advantages of this approach are that large numbers of mM ouabain, respectively. mutant genes are made available for analysis while the A retroviral shuttle vector containing the E. coli gpt gene amount of mammalian cell culture is minimized. However, (hereafter called pZip-GptNeo) was constructed by inserting shuttle-vector molecules spontaneously acquire unusually the gpt gene into the pZip-NeoSV(X)1 vector, which was high frequencies ofbase substitution, deletion, and insertion provided by C. Cepko. Plasmid construction was performed mutations when passaged through mammalian cells. according to standard procedures (14). Here we describe a somewhat different approach that has For DNA transfections, 60-mm culture dishes were inoc- enabled us to efficiently recover mutant genes from mam- ulated with 4 x 105 psi-2 cells. After 24 hr, the cells were malian cells while avoiding the problem of high spontaneous treated with vector DNA by the calcium phosphate method mutation frequencies. We have introduced a selectable gene (15). Four hours later, the DNA was removed and the cells into mammalian cells as part of a retroviral shuttle vector and were given a 20% (vol/vol) glycerol shock for 4 min. After 2 isolated infected cells that express the gene. After mutagen- days of growth under nonselective conditions, the cells from esis, cell lines with mutations in the selectable gene were each dish were harvested and plated in four 100-mm dishes isolated. Shuttle-vector sequences containing the mutant containing G418 medium. gene were then recovered from the mutant cell lines and For virus infections, G418r psi-2 colonies that had been introduced into E. coli. In contrast to earlier studies, the transfected with the pZip-GptNeo vector were pooled and population of shuttle-vector molecules used to transform E. coli is recovered from a clonal population of mutant cells. Abbreviations: bp, base pair(s); EtMes, ethyl methanesulfonate; This increases the likelihood that the recovered mutant gene HAT, hypoxanthine/aminopterin/thymidine; HPRTase, hypoxan- thine (guanine) phosphoribosyltransferase; kb, kilobase(s); LTR, long terminal repeat; MK, minimal agar medium with kanamycin; The publication costs of this article were defrayed in part by page charge MKSgu, minimal agar medium with kanamycin and thioguaniine; payment. This article must therefore be hereby marked "advertisement" SV40, simian virus 40; Sgu, 6-thioguanine; XPRTase, xanthine in accordance with 18 U.S.C. §1734 solely to indicate this fact. (guanine) phosphoribosyltransferase. 3356 Downloaded by guest on September 28, 2021 Genetics: Ashman et al. Proc. Natl. Acad. Sci. USA 83 (1986) 3357 grown to near confluence in a 100-mm dish. The G418 from a Moloney murine leukemia virus (Mo-MuLV) provirus medium was removed and replaced with nonselective medi- cloned in pBR322. Most of the viral sequences were deleted um (6 ml per dish). Twenty-four hours later, this medium was and unique BamHI and Xho I sites were created. The viral collected and filtered through 0.45-,um filters, and Polybrene sequences remaining include the long terminal repeats (8 ug/ml) was added to the filtrate. The filtrate was added to (LTRs), which contain sequences necessary for initiation and 60-mm dishes containing 5 x 105 A9 cells for 3 hr. Two days polyadenylylation of viral transcripts as well as integration of later, the cells were harvested and plated in either G418 or viral sequences into chromosomal DNA. Also, the vector HAT medium. contains the sequences necessary for reverse transcription of Recovery and Analysis of Vector DNA. Equal numbers (1.5 the viral genome and encapsidation ofviral RNA. To give this x 106) of virus-infected, HATr A9 cells and COS cells were vector the properties of a "shuttle" vector, a fragment plated together in 100-mm dishes and were fused 2 days later containing the simian virus 40 (SV40) origin of replication, by addition of 50% PEG plus 10% dimethyl sulfoxide for 1 the pBR322 origin of replication, and the neo gene from TnS min (16). Two days later, low molecular weight DNA was was introduced into the Xho I site. The presence of the SV40 extracted (17) and used to transform E. coli (18). Bacterial and pBR322 origins of replication enable the vector to strains DH-1 (18) and DT-2 (5) were provided by R. replicate in mammalian cells and bacteria, while the neo gene Kucherlapati (University of Illinois at Chicago). Kanamycin- provides a selectable marker in bacteria (Kanr) and mamma- resistant (Kan9 transformants were selected on plates con- lian cells (G418D. taining that drug at 50 ,g/ml. Sgur transformants were High titers of virus are produced when the pZip- selected as described (5). Preparation of plasmid DNA, NeoSV(X)l vector is introduced into psi-2 cells (11). This cell restriction enzyme digestion, agarose gel electrophoresis, line, which contains a defective Mo-MuLV provirus, supplies and Southern blotting were carried out as described (5). all the functions in trans necessary for the packaging ofRNA Mutagenesis. Virus-infected A9 cells maintained in HAT into virus but is incapable of packaging its own RNA. To medium were inoculated at a density of 106 cells per dish into determine whether the pZip-GptNeo vector could likewise be 150-mm tissue culture dishes containing medium supplement- packaged into a transmissible virus, it was transfected into ed with 0.1 mM hypoxanthine, 16 AM thymidine, and ethyl the psi-2 cell line and transformants were selected in G418 methanesulfonate (EtMes). After 18 hr, the medium was medium. In one experiment, the transfection of 2 x 106 psi-2 replaced with fresh G418 medium. After 10 days of growth in cells with 10 ,ug of vector DNA gave about 100 G418r G418 medium, the cells were harvested and mutant frequen- colonies. cies were determined. For Sgu resistance, three 100-mm Medium from cultures of G418r psi-2 cells was collected dishes were inoculated with 1-2 x 105 cells per dish; for and used as a source of virus to infect A9 cells.
Load more

Recommended publications
  • DNA Repair and Synthetic Lethality
    Int J Oral Sci (2011) 3:176-179. www.ijos.org.cn REVIEW DNA repair and synthetic lethality Gong-she Guo1, Feng-mei Zhang1, Rui-jie Gao2, Robert Delsite4, Zhi-hui Feng1,3*, Simon N. Powell4* 1School of Public Health, Shandong University, Jinan 250012, China; 2Second People’s Hospital of Weifang, Weifang 261041, China; 3Department of Radiation Oncology, Washington University in St Louis, St Louis MO 63108, USA; 4Department of Radiation Oncology, Memorial Sloan Kettering Cancer Center, New York NY 10065, USA Tumors often have DNA repair defects, suggesting additional inhibition of other DNA repair pathways in tumors may lead to synthetic lethality. Accumulating data demonstrate that DNA repair-defective tumors, in particular homologous recombination (HR), are highly sensitive to DNA-damaging agents. Thus, HR-defective tumors exhibit potential vulnerability to the synthetic lethality approach, which may lead to new therapeutic strategies. It is well known that poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP) inhibitors show the synthetically lethal effect in tumors defective in BRCA1 or BRCA2 genes encoded proteins that are required for efficient HR. In this review, we summarize the strategies of targeting DNA repair pathways and other DNA metabolic functions to cause synthetic lethality in HR-defective tumor cells. Keywords: DNA repair; homologous recombination; synthetic lethality; BRCA; Rad52 International Journal of Oral Science (2011) 3: 176-179. doi: 10.4248/IJOS11064 Introduction polymerase (PARP) inhibitors are specifically toxic to homologous recombination (HR)-defective cells [3-4]. Synthetic lethality is defined as a genetic combination Playing a key role in maintaining genetic stability, HR is of mutations in two or more genes that leads to cell a major repair pathway for double-strand breaks (DSBs) death, whereas a mutation in any one of the genes does utilizing undamaged homologous DNA sequence [5-6].
    [Show full text]
  • A Gain-Of-Function P53-Mutant Oncogene Promotes Cell Fate Plasticity and Myeloid Leukemia Through the Pluripotency Factor FOXH1
    Published OnlineFirst May 8, 2019; DOI: 10.1158/2159-8290.CD-18-1391 RESEARCH ARTICLE A Gain-of-Function p53-Mutant Oncogene Promotes Cell Fate Plasticity and Myeloid Leukemia through the Pluripotency Factor FOXH1 Evangelia Loizou1,2, Ana Banito1, Geulah Livshits1, Yu-Jui Ho1, Richard P. Koche3, Francisco J. Sánchez-Rivera1, Allison Mayle1, Chi-Chao Chen1, Savvas Kinalis4, Frederik O. Bagger4,5, Edward R. Kastenhuber1,6, Benjamin H. Durham7, and Scott W. Lowe1,8 Downloaded from cancerdiscovery.aacrjournals.org on September 27, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst May 8, 2019; DOI: 10.1158/2159-8290.CD-18-1391 ABSTRACT Mutations in the TP53 tumor suppressor gene are common in many cancer types, including the acute myeloid leukemia (AML) subtype known as complex karyotype AML (CK-AML). Here, we identify a gain-of-function (GOF) Trp53 mutation that accelerates CK-AML initiation beyond p53 loss and, surprisingly, is required for disease maintenance. The Trp53 R172H muta- tion (TP53 R175H in humans) exhibits a neomorphic function by promoting aberrant self-renewal in leu- kemic cells, a phenotype that is present in hematopoietic stem and progenitor cells (HSPC) even prior to their transformation. We identify FOXH1 as a critical mediator of mutant p53 function that binds to and regulates stem cell–associated genes and transcriptional programs. Our results identify a context where mutant p53 acts as a bona fi de oncogene that contributes to the pathogenesis of CK-AML and suggests a common biological theme for TP53 GOF in cancer. SIGNIFICANCE: Our study demonstrates how a GOF p53 mutant can hijack an embryonic transcrip- tion factor to promote aberrant self-renewal.
    [Show full text]
  • 701.Full.Pdf
    THE AVERAGE NUMBER OF GENERATIONS UNTIL EXTINCTION OF AN INDIVIDUAL MUTANT GENE IN A FINITE POPULATION MOT00 KIMURA' AND TOMOKO OHTA Department of Biology, Princeton University and National Institute of Genetics, Mishima, Japan Received June 13, 1969 AS pointed out by FISHER(1930), a majority of mutant genes which appear in natural populations are lost by chance within a small number of genera- tions. For example, if the mutant gene is selectively neutral, the probability is about 0.79 that it is lost from the population during the first 7 generations. With one percent selective advantage, this probability becomes about 0.78, namely, it changes very little. In general, the probability of loss in early generations due to random sampling of gametes is very high. The question which naturally follows is how long does it take, on the average, for a single mutant gene to become lost from the population, if we exclude the cases in which it is eventually fixed (established) in the population. In the present paper, we will derive some approximation formulas which are useful to answer this question, based on the theory of KIMURAand OHTA(1969). Also, we will report the results of Monte Carlo experiments performed to check the validity of the approximation formulas. APPROXIMATION FORMULAS BASED ON DIFFUSION MODELS Let us consider a diploid population, and denote by N and Ne, respectively, its actual and effective sizes. The following treatment is based on the diffusion models of population genetics (cf. KIMURA1964). As shown by KIMURAand OHTA (1969), if p is the initial frequency of the mutant gene, the average number of generations until loss of the mutant gene (excluding the cases of its eventual fixation) is - In this formula, 1 On leave from the National Institute of Genetics, Mishima, Japan.
    [Show full text]
  • Statement by the Group of Chief Scientific Advisors
    Statement by the Group of Chief Scientific Advisors A Scientific Perspective on the Regulatory Status of Products Derived from Gene Editing and the Implications for the GMO Directive On 25 July 2018, the Court of Justice of the European Biotechnology’ (SAM, 2017a), we have examined the Union ('the Court') decided that organisms obtained GMO Directive taking into account current by the new techniques of directed mutagenesis are knowledge and scientific evidence. genetically modified organisms (GMOs), within the meaning of the Directive 2001/18/EC on the release 1. The Ruling of the Court of Justice of genetically modified organisms into the On request by the French Conseil d'État, the Court environment ('GMO Directive')1,2, and that they are was asked to determine whether organisms subject to the obligations laid down by the GMO obtained by mutagenesis4 should be considered Directive. GMOs and which of those organisms are exempt New techniques of directed mutagenesis include according to the provisions of the GMO Directive. In gene editing such as CRISPR/Cas9 methodologies. particular, the Court was asked to determine The legal status of the products of such techniques whether organisms obtained by new directed was uncertain, because it was unclear whether they mutagenesis techniques are exempt from the fell within the scope of the GMO Directive. obligations imposed by the GMO Directive, as are those obtained by conventional, random These techniques enable the development of a wide mutagenesis techniques that existed before the range of agricultural applications and the ethical, adoption of the Directive, or are regulated like those legal, social and economic issues of their use are obtained by established techniques of genetic discussed intensively.
    [Show full text]
  • An Activated Rac Mutant Functions As a Dominant Negative for Membrane Ru‚Ing
    Oncogene (1998) 17, 625 ± 629 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc An activated Rac mutant functions as a dominant negative for membrane ruing Martin Alexander Schwartz, Jere E Meredith and William B Kiosses Department of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA Previous studies have shown that point mutations in the terminal eector domains (Westwick et al., 1997; eector domain of Rac1 block speci®c downstream Lamarche et al., 1996). Point mutants were con- pathways such as PAK, JNK/SAPK kinases and structed in V12 or Q61L backgrounds to give membrane ruing. Speci®cally, the F37A mutation, constitutively activated proteins that would show made in a constitutively activated Q61L background, dominant positive eects. These studies of Rac point activates PAK but fails to induce membrane rues. We mutations revealed that induction of membrane ruing now show that Q61L/F37A Rac not only fails to induce and cell cycle progression were independent of ruing but potently blocks membrane ruing induced by activation of PAK and other CRIB domain proteins, serum or PDGF. In the presence of serum, cells do and activation of the JNK/SAPK pathway (Westwick extend ®lopodia, suggesting that this mutant only blocks et al., 1997; Lamarche et al., 1996). a subset of the eectors that induce cytoskeletal One of these mutations, Q61L/F37A, was found to reorganization. At later times, this rac mutant induces abolish induction of membrane ruing and DNA membrane blebbing, but not apoptosis. These results synthesis but did not aect PAK or JNK/SAPK show that Q61L/F37A Rac, is constitutively activated activation (Lamarche et al., 1996).
    [Show full text]
  • Glossary/Index
    Glossary 03/08/2004 9:58 AM Page 119 GLOSSARY/INDEX The numbers after each term represent the chapter in which it first appears. additive 2 allele 2 When an allele’s contribution to the variation in a One of two or more alternative forms of a gene; a single phenotype is separately measurable; the independent allele for each gene is inherited separately from each effects of alleles “add up.” Antonym of nonadditive. parent. ADHD/ADD 6 Alzheimer’s disease 5 Attention Deficit Hyperactivity Disorder/Attention A medical disorder causing the loss of memory, rea- Deficit Disorder. Neurobehavioral disorders character- soning, and language abilities. Protein residues called ized by an attention span or ability to concentrate that is plaques and tangles build up and interfere with brain less than expected for a person's age. With ADHD, there function. This disorder usually first appears in persons also is age-inappropriate hyperactivity, impulsive over age sixty-five. Compare to early-onset Alzheimer’s. behavior or lack of inhibition. There are several types of ADHD: a predominantly inattentive subtype, a predomi- amino acids 2 nantly hyperactive-impulsive subtype, and a combined Molecules that are combined to form proteins. subtype. The condition can be cognitive alone or both The sequence of amino acids in a protein, and hence pro- cognitive and behavioral. tein function, is determined by the genetic code. adoption study 4 amnesia 5 A type of research focused on families that include one Loss of memory, temporary or permanent, that can result or more children raised by persons other than their from brain injury, illness, or trauma.
    [Show full text]
  • Genetic Engineering (3500 Words)
    Genetic Engineering (3500 words) Biology Also known as: biotechnology, gene splicing, recombinant DNA technology Anatomy or system affected: All Specialties and related fields: Alternative medicine, biochemistry, biotechnology, dermatology, embryology, ethics, forensic medicine, genetics, pharmacology, preventive medicine Definition: Genetic engineering, recombinant DNA technology and biotechnology – the buzz words you may have heard often on radio or TV, or read about in featured articles in newspapers or popular magazines. It is a set of techniques that are used to achieve one or more of three goals: to reveal the complex processes of how genes are inherited and expressed, to provide better understanding and effective treatment for various diseases, (particularly genetic disorders) and to generate economic benefits which include improved plants and animals for agriculture, and efficient production of valuable biopharmaceuticals. The characteristics of genetic engineering possess both vast promise and potential threat to human kind. It is an understatement to say that genetic engineering will revolutionize the medicine and agriculture in the 21st future. As this technology unleashes its power to impact our daily life, it will also bring challenges to our ethical system and religious beliefs. Key terms: GENETIC ENGINEERING: the collection of a wide array of techniques that alter the genetic constitution of cells or individuals by selective removal, insertion, or modification of individual genes or gene sets GENE CLONING: the development
    [Show full text]
  • Glossary of Common Terms in Genetics
    Glossary of Common Terms in Genetics Acquired mutations Gene changes genetic information. DNA is held Multiplexing A sequencing approach that that arise within individual cells and together by weak bonds between base uses several pooled samples simultaneous­ accumulate throughout a person's life pairs of nucleotides: adenine, guanine, ly, greatly increasing sequencing speed. span. cytosine, and thymine. Mutation Any heritable change in DNA Alleles One of a group of genes that Gene The fundamental unit of heredi­ sequence. occur alternatively at a given locus. A ty. A gene is an ordered sequence of single allele is inherited separately from nucleotides located in a particular posi­ Nucleotide A subunit of DNA or RNA each parent (e.g., at a locus for eye tion on a particular chromosome that consisting of a nitrogenous base, a phos­ color, the allele might result in blue or encodes a specific functional product phate molecule, and a sugar molecule. brown eyes). (i.e., a protein or RNA molecule i. Thousands of nucleotides are linked to form a DNA or RNA molecule. Base pair Two nitrogenous bases (ade­ Gene expression The process by which nine and thymine or guanine and cyto- a gene's coded information is converted Oncogene One or more forms of a sine) held together by weak bonds. Two into the structures present and operat­ gene associated with cancer. strands of DNA are held together in the ing in the cell. shape of a double helix by the bonds Polygenic disorders Genetic disorders between base pairs. Gene mapping Determination of the resulting from the combined action of relative positions of genes on a DNA alleles of more than one gene (e.g., Carrier A person who has a recessive molecule and the distance between heart disease, diabetes, and some can­ mutated gene along with its normal them.
    [Show full text]
  • The Qa Repressor Gene of Neurospora Crassa: Wild-Type and Mutant Nucleotide Sequences
    Proc. Natl. Acad. Sci. USA Vol. 83, pp. 3381-3385, May 1986 Genetics The qa repressor gene of Neurospora crassa: Wild-type and mutant nucleotide sequences (repressor protein/repressor mutants/eukaryotic gene regulation/gene cluster) LAYNE HuIET* AND NORMAN H. GILES Department of Genetics, University of Georgia, Athens, GA 30602 Contributed by Norman H. Giles, December 31, 1985 ABSTRACT The qa-JS gene, one of two regulatory genes The qa-JS genes from two constitutive mutants have also in the qa gene cluster of Neurospora crassa, encodes the qa been sequenced, revealing that the mutations are located in repressor. The qa-iS gene together with the qa-iF gene, which the distal region of the gene and suggesting that the COOH- encodes the qa activator protein, control the expression of all terminal region of the repressor protein may be involved in seven qa genes, including those encoding the inducible enzymes binding to its target, which may be the activator protein (1). responsible for the utilization of quinic acid as a carbon source. The qa-JS genes from two noninducible mutants had amino The nucleotide sequence of the qa-IS gene and its flanking acid substitutions just upstream of the constitutive muta- regions has been determined. The deduced coding sequence for tions, possibly implicating this region in binding the inducer. the qa-1S protein encodes 918 amino acids with a calculated Sequence analysis of these mutations also revealed a number molecular weight of 100,650 and is interrupted by a single of polymorphisms, primarily in the noncoding region of this 66-base-pair intervening sequence.
    [Show full text]
  • Isolation and Characterization of Mutant Cell Lines Defective in Transforming Growth Factor (3 Signaling BARBARA A
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7655-7660, July 1996 Cell Biology Isolation and characterization of mutant cell lines defective in transforming growth factor (3 signaling BARBARA A. HOCEVAR AND PHILIP H. HowE* Department of Cell Biology, Cleveland Clinic Research Institute, Cleveland, OH 44195 Communicated by George R Stark The Cleveland Clinic Foundation, Cleveland, OH, April 12, 1996 (received for review November 1, 1995) ABSTRACT To isolate and characterize effector mole- is thus formed, which initiates a downstream signaling cascade cules of the transforming growth factor 13 (TGF.8) signaling (10, 11). pathway we have used a genetic approach involving the Recent cloning of the receptors for the other members of the generation of stable recessive mutants, defective in their TGF superfamily reveals many interesting parallels with the TGFf3 signaling, which can subsequently be functionally com- TGF/3 system. For activin signaling, as for TGF,3 signaling, the plemented to clone the affected genes. We have generated a cell type I receptor requires the type II receptor to bind ligand line derived from a hypoxanthine-guanine phosphoribosyl- followed by the formation of a heteromeric complex that transferase negative (HPRT-) HT1080 clone that contains the initiates signaling (12). The type I receptors for the bone selectable marker Escherichia coli guanine phosphoribosyl- morphogenetic proteins and the decapentaplegic gene product transferase (gpt) linked to a TGFI8-responsive promoter. This are capable of binding ligand on their own; however, it appears cell line proliferates or dies in the appropriate selection that signaling in these systems also involves complex formation medium in response to TGFI3.
    [Show full text]
  • The Genetical Analysis of an Induced Height Mutant In
    Heredity (1980), 45(1),61-71 0018-067X/80/02040061$02.00 1980. The Genetical Society of Great Britain THEGENETICAL ANALYSIS OF AN INDUCED HEIGHT MUTANT IN WHEAT A. J. WORLAND, C. N. LAW and A. SHAKOOR* Plant Breeding institute, Cambridge, England Received4.ii.80 SUMMARY Three semi-dwarf mutants were isolated after treating monosomics of homo- eologous group 2 of the tall wheat "Bersée" with ethyl methanesulphonate (EMS). Two of the mutant lines were shown to have a single identical mutated gene, designated Rht7, on chromosome 2A. Little evidence could be found for chromosomes hemizygous at the time of treatment with mutagens having an increased sensitivity to mutation. Lines carrying Rht7 were shown to have reduced yields so that this mutation will probably be of little use in the breeding of high yielding short-strawed wheats. 1. INTRODUCTION IN bread wheat, Triticum aestivum (2n =6x=42),it has been suggested by Tsunewaki and Heyne (1959) that the chances of detecting a recessive mutant could be increased by treating monosomic rather than euploid seeds. Many recessive genes would be expressed on a hemizygous chromosome but would be masked if a non-mutated homologous chromosome was present. Chromosomes in the hemizygous condition also appear to be more sensitive to mutagens. If this approach of mutating monosomics is to be used, it should be possible to further increase the chances of mutating genes for a certain character by treating monosomics known to carry genes for the character in question. For final plant height each of the chromosomes of homoeologous group 2 were shown to have marked effects since monosomes of this group in the variety Bersée were uniformly dwarfed (Law and Worland, 1973).
    [Show full text]
  • How Genetic Engineering Differs from Conventional Breeding Hybridization
    GENETIC ENGINEERING IS NOT AN EXTENSION OF CONVENTIONAL PLANT BREEDING; How genetic engineering differs from conventional breeding, hybridization, wide crosses and horizontal gene transfer by Michael K. Hansen, Ph.D. Research Associate Consumer Policy Institute/Consumers Union January, 2000 Genetic engineering is not just an extension of conventional breeding. In fact, it differs profoundly. As a general rule, conventional breeding develops new plant varieties by the process of selection, and seeks to achieve expression of genetic material which is already present within a species. (There are exceptions, which include species hybridization, wide crosses and horizontal gene transfer, but they are limited, and do not change the overall conclusion, as discussed later.) Conventional breeding employs processes that occur in nature, such as sexual and asexual reproduction. The product of conventional breeding emphasizes certain characteristics. However these characteristics are not new for the species. The characteristics have been present for millenia within the genetic potential of the species. Genetic engineering works primarily through insertion of genetic material, although gene insertion must also be followed up by selection. This insertion process does not occur in nature. A gene “gun”, a bacterial “truck” or a chemical or electrical treatment inserts the genetic material into the host plant cell and then, with the help of genetic elements in the construct, this genetic material inserts itself into the chromosomes of the host plant. Engineers must also insert a “promoter” gene from a virus as part of the package, to make the inserted gene express itself. This process alone, involving a gene gun or a comparable technique, and a promoter, is profoundly different from conventional breeding, even if the primary goal is only to insert genetic material from the same species.
    [Show full text]