Biomedical subjects

R C Mulligan

Publications and source records attributed to R C Mulligan.

At least 127 records · Page 7Linked to original sources

PubMed1986

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.

Animals↗

PubMed1986

Retrovirus-mediated transfer of the human glucocerebrosidase gene to Gaucher fibroblasts.

A recombinant retrovirus carrying complementary DNA containing the complete coding sequence for human glucocerebrosidase was constructed and used to infect mouse cells and human fibroblasts from a type 2 Gaucher patient. The cDNA stably integrated in the genome of the recipient cell. Infected cells made glucocerebrosidase-cross-reacting material, and the protein was glycosylated and contained the epitope(s) missing in the type 2 mutant glucocerebrosidase. This study demonstrates the feasibility of efficiently transferring the gene encoding glucocerebrosidase to Gaucher cells using retrovirus as vector.

Animals↗

PubMed1985

Individual adenovirus type 5 early region 1A gene products elicit distinct alterations of cellular morphology and gene expression.

Adenovirus early region 1A (E1A), which gives rise to three overlapping transcripts, was inserted into a murine leukemia virus-derived vector, and recombinant viruses were used to prepare permanent cell lines of NIH 3T3 cells containing DNA copies of the individual 13S, 12S, and 9S mRNAs. Integrated proviral copies of the recombinant genomes were rescued as bacterial plasmids from each of the cell lines, and the DNA sequence of E1A was demonstrated to be a precise copy of the individual transcripts. The DNA copies were shown to be expressed as part of the full-length retroviral transcript by S1 nuclease analysis, and the synthesis of their encoded polypeptides was demonstrated by immunoprecipitation. Those cell lines expressing the polypeptide encoded by the 13S transcript were shown to contain that function required for regulating the accumulation of mRNAs from adenovirus early genes by their ability to complement the adenovirus type 5 E1A deletion mutant dl312. Cell lines expressing polypeptides encoded by the 13S, 12S, and 9S transcripts showed characteristic alterations in morphology. Two-dimensional gel electrophoresis of total cellular protein derived from the three cell lines demonstrated that each E1A gene product elicits specific alterations in the patterns of proteins expressed. Studies of the expression of two specific genes, those encoding fibronectin and collagen type 1, indicated that the observed alteration in levels of the two proteins results from a reduction in RNA levels induced by E1A functions.

Adenoviruses, Human↗

PubMed1985

H-2Ld antigen encoded by a recombinant retrovirus genome is expressed on the surface of infected cells.

A recombinant murine retrovirus was constructed which contains, within its genome, a truncated version of the gene encoding the murine H-2Ld major histocompatibility antigen. The H-2Ld gene, which was inserted 3' of the env splice acceptor site in the recombinant retrovirus MSV-neo, lacked the 5' promoter and TATA sequences and the 3' transcription termination and polyadenylate addition sites of the normal H-2Ld gene. Transfection of the MSV-neo/H-2Ld plasmid (pLTV-11) into Y-2 cells resulted in the production of the transmissible recombinant retrovirus LTV-11. Cells infected with LTV-11 virus were resistant to the eucaryotic antibiotic G418 and expressed H-2Ld on the cell surface. These infected cells contained a viral RNA species which possessed both the H-2Ld and the neomycin resistance gene sequences but did not contain significant levels of the smaller H-2Ld-specific mRNA. The H-2Ld antigen expressed on the surface of infected cells functioned as a target for cytolytic T cells specific for the H-2Ld antigen.

Animals↗

PubMed1984

Construction and applications of a highly transmissible murine retrovirus shuttle vector.

We develop a murine retrovirus shuttle vector system for the efficient introduction of selectable and nonselectable DNA sequences into mammalian cells and recovery of the inserted sequences as molecular clones. Three protocols allow rapid recovery of vector DNA sequences from mammalian cells. Two of the methods rely on SV40 T-antigen-mediated replication of the vector sequences and yield thousands of bacterial transformants per 5 X 10(6) mammalian cells. The majority of plasmids recovered by all three protocols exhibited the proper structure and were as active as the parental vector in the generation of transmissible retrovirus genomes upon transfection of mammalian cells. One of the rescue methods, which relies on "onion skin" replication and excision of an integrated provirus from the host chromosome, enables facile recovery of the chromosomal site of proviral integration. The system was also used to generate, and then efficiently recover, a cDNA version of a genomic insert from the adenovirus E1A region.

Cell Transformation, Viral↗

PubMed1984

Eukaryotic chromosome transfer: linkage of the murine major histocompatibility complex to an inserted dominant selectable marker.

We have developed an approach for genetic analysis of the murine H-2 complex that has broad general applicability to the study of eukaryotic genome organization. We have used a retroviral vector to introduce a selectable marker into the mouse genome close to the major histocompatibility complex (MHC). Chromosomal segments containing large portions of the MHC from these donor cells have been transferred both to hamster and monkey cell recipients. The procedure involved the following steps. First, a murine cell line was multiply infected with a defective recombinant murine leukemia virus that contains the neomycin-resistance gene (a gene that confers resistance to G418). In this way, the neomycin-resistance gene was introduced at multiple sites in the mouse genome. Second, metaphase chromosomes, prepared from this infected cell population, were transferred to hamster cell recipients. Third, two G418-resistant transferents were identified that expressed murine H-2 antigens on their cell surface. These transferents were shown to contain a large segment of the murine MHC (H-2K and I regions) by DNA hybridization. The neomycin-resistance gene and the mouse MHC genes must be physically linked in these cells since they could be cotransferred from the hamster cells to monkey cells. Fourth, the murine cell carrying the neomycin-resistance gene near the MHC was identified from the original donor cell population. This cell will serve as a useful source of chromosome fragments for analysis of larger portions of the MHC. This series of steps can serve as a paradigm for the first steps in a detailed genetic analysis of any specific region of a mammalian genome to which one or more genes have already been mapped.

Animals↗

PubMed1984

Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.

In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recombinant retrovirus encoding preproPTH, and this recombinant retrovirus was used to infect GH4 rat pituitary cells. Clonal lines of the infected GH4 cells contained copies of the recombinant provirus stably integrated via the long terminal repeats, and the expected RNA transcripts of proviral DNA accumulated in the cytoplasm, although no infectious virus was produced. The infected cells synthesized and processed preproPTH appropriately and secreted PTH in response to thyrotropin-releasing hormone, a secretagogue for GH4 cells. Use of recombinant retrovirus permits the introduction of DNA encoding normal and mutant secreted proteins into a number of cell types specialized for secretion. Analysis of the fate of the resultant proteins will help define the specific molecular interactions involved in transmembrane transport and processing of precursor proteins.

Amino Acid Sequence↗

PubMed1984

High-efficiency gene transfer into mammalian cells: generation of helper-free recombinant retrovirus with broad mammalian host range.

We have constructed a chimeric retrovirus genome containing ecotropic gag-pol sequences from Moloney murine leukemia virus and envelope sequences derived from the amphotropic virus 4070A. This reconstructed genome, termed pMAV-psi-, lacks the psi site required for encapsidation of the viral genome. NIH 3T3 cells transfected with pMAV-psi-, called psi-AM lines, are capable of producing high titer stocks of helper-free recombinant retrovirus with amphotropic host range after transfection with recombinant retroviral vectors carrying the neomycin phosphotransferase gene. Most transfected psi-AM cells remain helper-free, even after months in culture. psi-AM virus stocks infect nearly all human and murine cell lines tested thus far, as assayed by resistance to the neomycin analogue G418. Southern and RNA blot analyses of psi-AM-infected human cells show that recombinant murine retroviruses integrate randomly into genomic DNA as normal proviruses and express high levels of the subgenomic and genomic viral messages in the expected stoichiometry of 1:1.

Animals↗

PubMed1984

Introduction of a selectable gene into different animal tissue by a retrovirus recombinant vector.

The potential use of retrovirus vectors to transduce foreign genetic information into cells of different tissues of an animal was explored by introducing a recombinant genome carrying the Eco gpt gene into postimplantation mouse embryos. To obviate the need for preparing concentrated virus stocks, psi 2-2-5 cells producing the replication-defective murine sarcoma virus (MSV)-gpt virus were microinjected directly into embryos. The psi 2-2-5 cells were mixed with cells producing replication-competent Moloney murine leukemia virus (Mo-MuLV) to facilitate spread of the vector. A high percentage of the manipulated embryos continued to develop without disturbance and were analyzed either prior to birth or as adults for expression of both helper and Eco gpt virus. Microinjection of as few as 10 Mo-MuLV-producing cells resulted in viremia of greater than 50% of the embryos or adults, 25%-30% of which produced MSV-gpt recombinant virus in a variety of organs including thymus, spleen, lung, kidney, and brain. The fraction of vector-producing cells, however, was 3 to 5 orders of magnitude lower than that of helper-virus-producing cells. Our results demonstrate that a selectable gene can be introduced by retroviral vectors into animals and can be expressed in a wide variety of different somatic tissues.

Animals↗

PubMed1984

Metaphase chromosome transfer of introduced selectable markers.

A general method for creating somatic cell hybrids which maintain chromosome segments by selection is described. To extend the technique of metaphase chromosome transfer to regions of the genome which do not carry a selectable marker, a retroviral vector has been employed to introduce the dominant selectable neo gene at many genomic locations in a population of murine cells. We have used such cells as donors in metaphase chromosome transfer experiments in which hamster and monkey cells were used as recipients. Cells acquiring a transferred chromosomal segment containing the neo gene were selected by growth in the presence of the drug G418. Hybridization to cloned interspersed repeat DNA sequences of the mouse was used to estimate the proportion of mouse DNA in each transferent. These experiments indicate that transferents produced in this manner contain 0.01 to 1.0% of the mouse genome. To analyze the organization of the DNA transferred to each recipient, we used Southern transfer hybridization of DNA from each transferent to a cloned mouse interspersed repeated DNA probe which did not cross-hybridize to hamster or monkey DNA. We found that each primary transferent gave a unique pattern of restriction fragments hybridizing to this probe. Secondary transferents from two independent primary transferents were compared by this technique. Each set of secondary transferents exhibited a hybridization pattern which resembled closely that of the primary transferent from which it was derived. However, a number of hybridizing DNA segments present in each primary transferent were absent in some of the secondary transferents. These results are most compatible with the view that an intact segment of the mouse chromosome surrounding the integration site of the retroviral vector can be transferred by the techniques used in this study. We believe this technique will be generally applicable to cells from many species, and will allow the study of chromosome regions previously refractory to analysis by chromosome transfer techniques.

Animals↗

PubMed1983

Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus.

A mutant of Moloney murine leukemia virus (M-MuLV), pMOV-psi-, was constructed by deletion of about 350 nucleotides from an infectious proviral DNA clone between the putative env mRNA 5' splice site and the AUG that initiates the coding sequence for Pr65gag. Although the parent wild-type proviral clone, pMOV-psi+, quickly causes a high level of reverse-transcriptase-containing virus particles to be released from transfected NIH/3T3 cells, transfection of pMOV-psi- into these cells initially results in very little release. By 9 to 10 days after transfection, however, pMOV-psi- -transfected cells produce infectious virus. Thus pMOV-psi- has a defect that can be repaired in transfected NIH/3T3 cells, presumably by recombination with a sequence normally present in the cells. Cell lines with pMOV-psi- stably integrated into chromosomal DNA produce reverse-transcriptase-containing particles that lack detectable M-MuLV RNA but the cells efficiently complement replication-defective, packagable retroviruses. Thus pMOV-psi- has a defect in the packaging of genomic RNA into virions but can provide in trans the products necessary for virion production. The deletion in pMOV-psi- appears to define a site required in cis for packaging of MuLV RNA into virions. Cell lines carrying pMOV-psi- can be used to produce helper-free stocks of natural or synthetic defective retroviruses.

Chromosome Deletion↗

PubMed1981

Selection for animal cells that express the Escherichia coli gene coding for xanthine-guanine phosphoribosyltransferase.

Cultured monkey (TC7) and mouse (3T6) cells synthesize an Excherichia coli enzyme, xanthine-guanine phosphoribosyltransferase (XGPRT; 5-phospho-alpha-D-ribose-1-diphosphate:xanthine phosphoribosyltransferase, EC 2.4.2.22), after transfection with DNA vectors carrying the corresponding bacterial gene, Ecogpt. In contrast to mammalian cells, which do not efficiently use xanthine for purine nucleotide synthesis, cells that produce E. coli XGPRT can synthesize GMP from xanthine via XMP. After transfection with vector-Ecogpt DNAs, surviving cells producing XGPRT can be selectively grown with xanthine as the sole precursor for guanine nucleotide formation in a medium containing inhibitors (aminopterin and mycophenolic acid) that block de novo purine nucleotide synthesis. Cells transformed for Ecogpt arise with a frequency of 10(-4) to 10(-5); they appear to be genetically stable in as much as there is no discernible decrease in XGPRT formation or loss on their ability to grow in selective medium after propagation in nonselective medium. Although several of the vector-gpt DNAs can replicate in monkey and mouse cells, none of the transformants contain autonomously replicating vector-gpt DNA. Rather, the gpt transformants contain one to five copies of the transfecting DNA associated with, and most probably integrated into, cellular DNA sequences. In several transformants, vector-coded gene products for which there was no selection are also synthesized. This suggests that recombinant DNAs containing Ecogpt as a selective marker can be useful for cotransformation of nonselectable genes.

Animals↗

PubMed1981

Factors governing the expression of a bacterial gene in mammalian cells.

Cultured monkey kidney cells transfected with simian virus 40 (SV40)-pBR322-derived deoxyribonucleic acid (DNA) vectors containing the Escherichia coli gene (Ecogpt, or gpt) coding for the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT) synthesize the bacterial enzyme. This paper describes the structure of the messenger ribonucleic acids (mRNA's) formed during the expression of gpt and an unexpected feature of the nucleotide sequence in the gpt DNA segment. Analyses of the gpt-specific mRNA's produced during infection of CV1 cells indicate that in addition to the mRNA's expected on the basis of known simian virus 40 RNA splicing patterns, there is a novel SV40-gpt hybrid mRNA. The novel mRNA contains an SV40 leader segment spliced to RNA sequences transcribed from the bacterial DNA segment. The sequence of the 5'-proximal 345 nucleotides of the gpt DNA segment indicates that the only open translation phase begins with an AUG about 200 nucleotides from the end of the gpt DNA. Two additional AUGs as well as translation terminator codons in all three phases precede the XGPRT initiator codon. Deletion of the two that are upstream of the putative start codon increases the level of XGPRT production in transfected cells; deletion of sequences that contain the proposed XGPRT initiator AUG abolishes enzyme production. Based on the location of the XGPRT coding sequence in the recombinants and the structure of the mRNA's, we infer that the bacterial enzyme can be translated from an initiator AUG that is 400 to 800 nucleotides from the 5' terminus of the mRNA and preceded by two to six AUG triplets.

Animals↗

PubMed1980

Expression of a bacterial gene in mammalian cells.

Transfection of cultured monkey kidney cells with recombinant DNA constructed with a cloned Escherichia coli gene that codes for xanthine-guanine phosphoribosyltransferase and several different SV40 DNA-based vectors, results in the synthesis of readily measurable quantities of the bacterial enzyme. Moreover, the physiological defect in purine nucleotide synthesis characteristic of human Lesch-Nyhan cells can be overcome by the introduction of the bacterial gene into these cells.

Animals↗

PubMed1979

Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome.

Rabbit beta-globin complementary DNA (cDNA) has been inserted into SV40 DNA in place of the gene coding for the virus' major capsid protein, VP1. The recombinant genome, SVGT5-RabetaG, multiplies efficiently in CV1 monkey kidney cell cultures and is transcribed to yield cytoplasmic, polyadenylated hybrid mRNAs containing the beta-globin coding sequence. Cells propagating SVGT5-RabetaG produce substantial quantities of rabbit beta-globin polypeptide.

Animals↗

PubMed1976

Direct biochemical mapping of eukaryotic viral DNA by means of a linked transcription-translation cell-free system.

A method is described for mapping regions of eukaryotic viral DNA coding for specific proteins, utilizing a linked transcription-translation cell-free system primed with DNA fragments generated by restriction endonucleases. Three simian virus 40 (SV40) DNA fragments derived from that region of the DNA expressed late in lytic infection were purified. They were: Hpa I-A (0.76-0.175 map units), Bgl I-EcoRI-B (0.672-0), and Hpa II-EcoRI-B (0.735-0). (Fragments are named from the cleaving restriction endonuclease and electrophoretic mobility. End positions on the conventional map are in clockwise order.) These fragments efficiently stimulated the incorporation of [3H]UTP and [35S]methionine into trichloroacetic-acid-insoluble material in the linkec system. The location of the region of DNA coding for the viral structural proteins VPI, VP2 and VP3 was determined from the spectrum of polypeptide synthesis directed by the individual intact fragments and their specific endonucleolytic digests. The polypeptides synthesized in the cell-free system were characterized on urea-sodium dodecyl sulfate polyacrylamide gradient gels and by two-dimensional tryptic peptide analysis. ..

Cell-Free System↗

PubMed1975

Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system.

A linked cell-free system has been developed which is capable of transcribing and translating mamalian viral DNA, and its characteristics and requirements are outlined. In this system, simian virus 40 (SV40) DNA Form I (supercoiled) directed the synthesis of discrete polypeptides up to 85,000 daltons in size. One of these products was indistingusihable from authentic major virus capsid protein VPI, as judged by mobility on sodium dodecyl sulfate/polyacrylamide gels, antibody predipitation, and peptide analyses. The cell-free products larger than VPI comprised a number of polypeptides ranging in molecular weight from 50,000 to 85,000. These polypeptides demonstrated no immunological relationship whatsoever to the structural protein VPI. However, two of these products, along with one of approximately 25,000 dlatons, were precipitated with antiserum to SV40 tumor antigen. Linear SV40 DNA generated by the cleavage of Form I DNA with the restriction endonuclease EcoR1 was an efficient template in this system and also directed the synthesis of a polypeptide migrating with VPI on polyacrylamide gels. The potential of this system for defining a functional map of a DNA genome is discussed.

Animals↗

PubMed1974

Pre-proparathyroid hormone: a direct translation product of parathyroid messenger RNA.

An 8-15S RNA fraction from calf parathyroid glands stimulated the incorporation of radioactive lysine and methionine into protein by 15- to 30-fold in a wheat germ extract. The major product, representing 25% of the total protein synthesized, could be bound to an antiserum to parathyroid hormone and binding was inhibited by parathyroid hormone. The chromatographic mobilities of the two tryptic peptides of the cell-free product that contained methionine were identical to the corresponding peptides of parathyroid hormone. Upon electrophoresis in acidic or sodium dodecyl sulfate-acrylamide gels, the cell-free product migrated more slowly than either parathyroid hormone or its biosynthetic precursor, proparathyroid hormone. Analysis of cyanogen bromide products indicated that the cell-free product contained an additional sequence of amino acids at the amino-terminal end. A protein corresponding to the cell-free product could not be detected in intact cells even during incubations with [(3)H]leucine as short as 2 min, which suggests the protein may be a transient precursor to proparathyroid hormone.

Amino Acid Sequence↗