Mouse model for somatic mutation at the HPRT gene: molecular and cellular analyses.
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Animals↗
Biomedical subjects
I M Jones
Publications and source records attributed to I M Jones.
The protease and gag gene products of the human immunodeficiency virus: authentic cleavage and post-translational modification in an insect cell expression system.
Three recombinant baculoviruses which are capable of expressing human immunodeficiency virus (HIV) protease, p55gag, and both products simultaneously in insect cell culture have been constructed. Upon co-infection of cells with the protease and p55gag-expressing viruses, authentic processing of the gag precursor is observed to take place. This processing could be reproduced in vitro using mixtures of cellular lysates containing the expressed proteins. When expressed alone, uncleaved p55gag precursor appears to form retroviral core-like particles within the cytoplasm of infected cells. Metabolic labeling studies of the baculovirus-expressed gag products have demonstrated that p17 is myristylated at its amino terminus, and that p24 is phosphorylated. In these respects, the insect cell system is evidently capable of carrying out post-translational processing resembling that which occurs in authentic HIV-1 replication.
Acylation↗
Recognition of the PB1, neuraminidase, and matrix proteins of influenza virus A/NT/60/68 by cytotoxic T lymphocytes.
We have investigated the recognition of the PB1, neuraminidase, and matrix (M1) proteins of influenza virus A/NT/60/68 (H3N2 subtype) by secondary in vitro stimulated polyclonal cytotoxic T lymphocyte (CTL) populations. While these three proteins have different functions and cellular locations, they can all be recognized as target antigens. However, the immunogenicity of these proteins for CTLs is under strict genetic control. Thus, PB1 protein is recognized as a cross-reactive target antigen by CTLs raised in CBA (H-2k) but not BALB/c (H-2d) mice. CBA, but not BALB/c mice, also generate a low-level CTL response to the neuraminidase. This latter response was only detectable following in vivo priming of CBA mice with a recombinant vaccinia virus expressing neuraminidase (N2-VACC). The matrix protein, expressed from recombinant vaccinia virus M-VACC, was not recognized as an antigen by CTL generated from either CBA or BALB/c strains of mice. By contrast, human HLA-A2-restricted influenza virus-specific CTLs were shown to recognize this matrix protein as a target antigen. Endogenous expression of as little as 90 amino acids of the matrix protein was sufficient to render target cells susceptible to lysis by such CTLs.
Animals↗
Cell proliferation in the bone marrow, thymus and spleen of mice studied by continuous, in vivo bromodeoxycytidine labelling and flow cytometric analysis.
We have applied the technique of labelling dividing cells with bromodeoxyuridine (BrdUrd) in combination with in vivo continuous labelling, propidium iodide (PI) staining for DNA content, and flow cytometric analysis, for the determination of cell proliferation in bone marrow, thymus and spleen of mice. The percentage of BrdUrd labelled cells increased as a function of exposure time in a tissue specific manner for each of the three tissues. Thymus and bone marrow had cell populations which exhibited different kinetics for the accumulation of label: (1) those that cycled and became labelled within 2-3 days (88% in 2 days for bone marrow, 84% in 3 days for thymus); (2) those that cycled during the remainder of the 6 day infusion period (11% of bone marrow and 13% of thymus cells); and (3) those that did not cycle during the 6 day period studied (less than 2% of bone marrow and 3% of thymus cells). In contrast, the spleen exhibited a slower, constant accumulation of labelled cells. After six days of infusion a large proportion of spleen cells (50%) had not become labelled. These results suggest that a larger proportion of spleen cells are long lived than indicated by other methods. We also have found the period of labelling with BrdUrd extended several days beyond the period of infusion. This method will be very useful in studying perturbations of cell populations induced in mice exposed to toxic agents.
Animals↗
Direct RNA binding by the rev product of HIV-1.
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Gene Products, rev↗
The role of beta-hydroxyaspartate and adjacent carboxylate residues in the first EGF domain of human factor IX.
beta-Hydroxyaspartic acid is a post-translationally modified amino acid found in a number of plasma proteins in a domain homologous to epidermal growth factor. Its presence can be correlated with a high affinity Ca2+ binding site, with a dissociation constant of 10-100 microM. We describe a system for the expression of human coagulation factor IX in dog kidney cells in tissue culture, in which the post-translational modifications and the biochemical activity are indistinguishable from factor IX synthesized in vivo. This system has been used to express eight different point mutations of human factor IX in the first epidermal growth factor domain in order to study the role of beta-hydroxyaspartate at residue 64, and the adjacent carboxylate residues at positions 47, 49 and 78. We conclude that this domain is essential for factor IX function and suggest that Ca2+ binds to carboxylate ions in this domain and stabilizes a conformation necessary for the interaction of factor IXa with factor X, factor VIII and phospholipid in the next step of the clotting cascade.
Amino Acid Sequence↗
A region of the influenza A/NT/60/68 PB2 protein containing an antigenic determinant recognized by murine H-2Dd restricted cytotoxic T lymphocytes.
We have used a recombinant vaccinia virus to investigate the recognition of the PB2 protein of influenza A/NT/60/68 (H3N2) by murine polyclonal CTL populations. PB2 is recognized as a major cross-reactive target antigen. Recognition of PB2 is under strict genetic control, since BALB/c (H-2d) but not CBA (H-2k) mice are responders. We also demonstrate, by use of cell lines transfected with individual genes encoding class I molecules of the H-2d haplotype, that recognition of PB2 occurs in conjunction with the H-2Dd but not the H-2Kd or H-2Ld molecules. In contrast, recognition of the nucleoprotein of A/PR/8/34 by BALB/c-derived polyclonal CTL is restricted via the H-2Kd molecule. By using three recombinant vaccinia viruses expressing deleted forms of the PB2 protein we show that at least one epitope of the PB2 protein resides within the amino-terminal 256 amino acids. This approach offers an effective method to map the regions of large proteins containing epitopes recognized by CTL.
Animals↗
Localization of RNA polymerases on influenza viral ribonucleoproteins by immunogold labeling.
Monospecific antisera for the influenza polymerase proteins and high resolution immunoelectron microscopy have been used to investigate the topographical distribution of the polymerase molecules on influenza ribonucleoproteins (RNPs). Antibodies to PB1, PB2, and PA identify a single polymerase binding site located at, or very close to, the end of each RNP. Double labeling experiments confirm that all three polymerases are at the same end of each RNP and that they are in close association.
DNA-Directed RNA Polymerases↗
Factors that affect the frequency of thioguanine-resistant lymphocytes in mice following exposure to ethylnitrosourea.
The frequency of thioguanine(TG)-resistant lymphocytes in mice treated with ethylnitrosourea (ENU) was followed for a period of 51 wk using our clonogenic assay [Jones et al, 1985a,b]. The effects of dose (0-58 mg/kg), time since treatment (2-51 wk), dose rate (5 weekly X 11.7 mg/kg versus 1 X 58 mg/kg), and age at time of treatment (3 vs 15 mo) on the frequency of TG-resistant, concanavalin A-responsive spleen cells were evaluated. The frequencies of TG-resistant spleen cells were generally dose responsive for 51 wk after exposure to ENU. They also were dependent upon the time that had elapsed since treatment with ENU, increasing to maximal values at 10 wk as previously reported [Jones et al, 1985a], and holding essentially stable at values of approximately 20% of the maximum frequency from week 15 until at least week 40 for the 3-month-old mice. Fractionation of 58 mg ENU/kg into 5 weekly doses did not affect the frequency of ENU-induced TG-resistant cells detected in the spleen but did increase the rate of appearance in the spleen, and the efficiency of induction by the unit dose, of TG-resistant cells. The mice exposed to ENU at 15 mo of age appeared to have a 4-fold reduction in the rate of increase in frequency of ENU-induced TG-resistant spleen cells. One set of control mice was found to have a 10-fold elevated frequency of TG-resistant cells in both the spleen and thymus, indicating that mutations can occur in stem cells of untreated animals.
Age Factors↗
Cloned mouse lymphocytes permit analysis of somatic mutations that occur in vivo.
As part of our mouse model of somatic mutation, we have begun to characterize spontaneously occurring hypoxanthine phosphoribosyltransferase (HPRT) -deficient mouse lymphocytes. Lymphocytes were cloned by in vitro exposure of spleen cells from male C57B1/6 mice to the mitogen concanavalin A, conditioned medium containing lymphocyte growth factors, and thioguanine (TG), in a limiting dilution assay. The 17 TG-resistant clones recovered were all highly deficient in HPRT activity and were found by analysis of surface antigens to be representative of the major subclasses of T lymphocytes. Southern analysis of lymphocyte genomic DNA detected alterations of the hprt gene in 12/17 of the HPRT-deficient lymphocyte clones. Of these 12, 2/17 were lacking the entire hprt locus, 7/17 lacked part of the locus, and 3/17 had other, unidentified alterations.
Animals↗
Assembly of influenza ribonucleoprotein in vitro using recombinant nucleoprotein.
The influenza A virus nucleoprotein previously expressed in Escherichia coli after fusion to 32 heterologous amino acids has now been purified and tested for its ability to form complexes with RNA in vitro. By using a simple filter binding assay, we show that ribonucleoprotein (RNP) complexes form readily with single-stranded RNA of viral or nonviral origin but not with double-stranded RNA. The RNP complexes formed were similar to authentic influenza virus RNPs in appearance under the electron microscope, in buoyant density in gradients of cesium chloride, and in sensitivities to pancreatic ribonuclease, to chaotropic reagents, and to high salt. We conclude that nucleoprotein synthesized in E. coli has all the properties required for correct assembly into ribonucleoprotein.
Binding, Competitive↗
Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2.
In order to re-examine the sub-cellular location of the three influenza A/NT/60/68 polymerase proteins PB1, PB2 and PA in infected cells, specific antisera for each polymerase component have been prepared by immunizing rabbits with polymerase-beta-galactosidase fusion proteins synthesized in Escherichia coli. We show that polymerase PB1, PB2, and PA are predominantly associated with the nucleus of influenza-infected MDCK cells by immunocytochemical techniques. In the case of polymerase PB2 we investigate the possibility that nuclear accumulation is an intrinsic property of the PB2 protein. Using a vaccinia-PB2 recombinant virus, we show that PB2 accumulates intra-nuclearly in monkey CV-1 cells in the absence of any other influenza protein, suggesting it contains an intrinsic nuclear signal.
Animals↗
Molecular cloning and nucleotide sequence of human alpha 1 acid glycoprotein cDNA.
A cDNA clone has been isolated from a pEX expression library that encodes alpha 1 acid glycoprotein. We present the complete nucleotide sequence encoding this protein and compare the derived amino acid sequence with pre-existing data.
Amino Acid Sequence↗
Autoradiographic detection of HPRT variants of human lymphocytes resistant to RNA synthesis inhibition.
The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.
Autoradiography↗
A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes.
A clonogenic assay to quantify thioguanine (TG)-resistant (TGr) spleen lymphocytes in the mouse has been developed to support studies of in vivo mutation affecting the hypoxanthine phosphoribosyltransferase (hprt) locus. Lymphocytes are cultured in 96-well microtiter plates for 9 days with proliferation initiated by the mitogen concanavalin A and supported thereafter by conditioned medium containing interleukin-2. Lymphocytes are plated at high densities (4-8 X 10(5)/well) with TG and irradiated L5178Y lymphoma cells (10(4)/well) to detect the presence of TGr cells. To determine the cloning efficiency without TG lymphocytes are plated at a low density (10/well) with irradiated L5178Y cells and irradiated lymphocytes (4-8 X 10(5)/well). Proliferation of cells is detected by [3H]thymidine incorporation and scintillation spectrometry. Spontaneous frequencies of TGr clones are independent of TG dose from 0.2 to 10 micrograms/ml and independent of cell density over the range cited. The TGr clones tested have less than 10% hypoxanthine incorporation in vivo relative to unselected clones and have stable phenotypes in the absence of selection. The spontaneous frequency of TGr cells ranged from 1 to 3 X 10(-6). In vivo treatment of mice intraperitoneally with ethylnitrosourea 15 days prior to in vitro culture resulted in a linear dose-related increase of TGr cells, with 70.2 mg/kg inducing a frequency of TGr cells of 2 X 10(-5).
Animals↗
A study of the frequency of sister-chromatid exchange and of thioguanine-resistant cells in mouse spleen lymphocytes after in vivo exposure to ethylnitrosourea.
As part of the development of a multi-endpoint, in vivo, mouse model for mutagenesis we have measured the frequency of sister-chromatid exchange (SCE) and the frequency of thioguanine-resistant (TGr) cells among the lymphocytes of the mouse spleen following acute, intraperitoneal exposure to ethylnitrosourea (ENU). The responses of these two endpoints have been monitored both as a function of the dose of ENU injected, ranging from 0 to 70 mg/kg, and as a function of time after injection, from 1 day to 72 days. The SCE frequency response was highest 1 day after the ENU was injected, increasing 2.5-fold over control values for mice that received 70 mg/kg, and declined to control values in all animals by 72 days. SCE showed a linear dose response both at 1 day and 8 days after injection. The frequency of TGr cells was at control levels at 1 day, but at 15, 36 and 72 days after ENU injection the frequency of TGr cells showed a linear dose response. In addition, the frequency of TGr cells increased linearly with time for both the 35 and 70 mg/kg doses. The frequency of TGr cells for mice that had received 70 mg ENU/kg 72 days previously, was 100-fold higher than in control animals, giving a frequency of 1.4 X 10(-4).
Animals↗
Differential expression of influenza N protein and neuraminidase antigenic determinants in Escherichia coli.
Two influenza gene products of similar size and codon usage have been expressed in Escherichia coli under control of the phage lambda pR promoter. The influenza N protein (NP) was expressed in its entirety after fusion to a short (12 amino acid) segment of the lambda cro gene product and constituted about 1-2% of total soluble cell protein after induction. By contrast, constructions using the full length neuraminidase (NA) gene failed to give rise to detectable amounts of NA antigen after fusion to either the 12 amino acid Cro peptide or after fusion to bacterial beta-galactosidase (beta gal). Rather, expression of NA antigenic determinants was only achieved after deletion of coding sequences at the 3' end of the beta gal-NA fusion construct such that the encoded protein precipitated within the cell.
Antigens, Viral↗
Recombination between short direct repeats in a recA host.
Spontaneous deletion derivatives of plasmid pHV14, a recombinant of pBR322, have been isolated in the recA strain HB101. About 2.5 kilobases (Kb) of DNA has been lost in each of the four plasmids described including, in two cases the region involved in control of copy number and the initiation of replication. Sequence analysis of each deletion junction has revealed that in 3 out of 4 cases the deletion event occurred by recombination between short direct repeats of as little as 7 base pairs (bp).
Adenosine Triphosphatases↗