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[65] Methods for mapping human interferon structural genes
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[65] M e t h o d s for M a p p i n g H u m a n I n t e r f e r o n Structural Genes By DORIS L. SLATE and FRANK H. RUDDLE
Studies on the human interferon system have revealed a complex set of genetic and epigenetic factors that influence the synthesis and action of interferon. Interferon induction requires de novo RNA and protein synthesis as determined through the use of transcription and translation inhibitors. 1 Cells that are actively synthesizing interferon can be enucleated, and the resulting cytoplasts can still produce interferon. 2 The yield of interferon can vary over a wide range for human cells, depending on such factors as the " a g e " of a culture, the inducing stimulus, and whether the cells have a limited life-span or are "transformed" to indefinite growth in culture. Not only do the cell type and induction regimen affect the amount of interferon made, they can also affect the type of interferon made. Three major types of human interferon have been described: fibroblast (F), leukocyte (Le), and immune (sometimes called T or type II) interferons. F type is usually produced in fibroblast cultures treated with poly(I) • poly(C); Le type is usually made in buffy coat cells or in virus-transformed established lymphoblastoid lines after viral induction. Immune interferon is produced by various cells of the immune system in response to several types of stimulation (for example, treatment with mitogens or exposure to antigens that trigger immune recognition). These human interferon species differ in a number of respects, including stability at pH 2 and high temperature, antigenically, in molecular weight, and in cross-species antiviral activity. Havell, Hayes, and Vil~ek~ have reported that human fibroblasts can synthesize both F and Le type interferons after viral induction. The human interferon system is also amenable to various treatments that can modulate the amount or time course of interferon synthesis. Two major examples are priming and superinduction. In priming, low doses of interferon are used to pretreat cells, which are then normally induced; interferon is produced earlier than in unprimed control cells treated with just the inducer. 4 Superinduction involves the use of suitably timed doses of translation and transcription inhibitors and induction with poly(I) 1 M. Ho and J. A. Armstrong, Annu, Rev. Microbiol. 29, 131 (1975). 2 D. C. Burke and G. Veomett, Proc. Natl. Acad. Sci. U.S.A. 74, 3391 (1977). 3 E. A. Havell, T. G. Hayes, and J. Vil~ek, Virology 89, 330 (1978). 4 W. E. Stewart, II, L. B. Gosser, and R. Z. Lockart, J. Virol. 7, 792 (1971).
METHODS IN ENZYMOLOGY, VOL, 79
Copyright ~ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979-5
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poly(C). This technique lengthens the period of interferon synthesis and increases final yields. On a molecular level, superinduction increases the amount of interferon messenger RNA synthesized and also stabilizes this messenger.5,6 The genetic control of human interferon synthesis and mechanism of action has been examined in tissue culture systems via somatic cell hybridization and the study of aneuploid or aneusomic human cell lines. We will attempt to summarize the major techniques and findings in the mapping of genes governing human interferon production. Somatic Cell Hybrids in Human Interferon Gene Mapping Most of the somatic cell genetic studies that have resulted in human gene assignments have been performed with human/rodent hybrid cells. Human/mouse and human/Chinese hamster hybrid cell lines tend to lose human chromosomes preferentially, making it possible to correlate the presence or the absence of a specific human phenotype with the presence or the absence of a particular human chromosome. We will not describe the generation of hybrids, or the isozyme and karyotype analysis, required for gene mapping. Detailed procedures for these techniques can be found in the literature (for example, see Kennett7 for a discussion of hybridization methods, Nichols and Ruddle s and Kozak et al. 9 for starch gel electrophoretic conditions to distinguish mouse and human isozymes, and Kozak, Lawrence, and Ruddle 1° and Worton and Duff11 for karyotype procedures for analyzing hybrid cells). The first assignment for human genes involved in the synthesis of interferon was made by Tan, Creagan, and Ruddle. 12They characterized a large number of human/mouse hybrid lines and found that the ability to produce human interferon could not be associated with the presence of any one human chromosome. When they examined the correlation between human interferon production and the presence of two human chromosomes together, however, they found that when a hybrid con5 p. B. Sehgal and I. Tamm, Virology 92, 240 (1979). 6 R. L. Cavalieri, E. A. Havell, J. Vil~ek, and S. Pestka, Prec. Natl. Acad. Sci. U.S.A. 74, 4415 (1977). R. H. Kennett, this series, Vol. 58, p. 345. s E. A. Nichols and F. H. Ruddle, J. Histochern. Cytochem. 21, 1066 (1973). 9 C. A. Kozak, R. E. K. Fournier, L. A. Leinwand, and F. H. Ruddle, Biochem. Genet. 17, 23 (1979). ao C. A. Kozak, J. B. Lawrence, and F. H. Ruddle, Exp. Cell Res. 105, 109 (1977). 1~ R. G. Worton and C. Duff, this series, Vol. 58, p. 322. Y. H. Tan, R. P. Creagan, and F. H. Ruddle, Prec. Natl. Acad. Sci. U.S.A. 71, 2251 (1974).
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tained both human chromosomes 2 and 5, human interferon synthesis could be induced with proper stimulation. They also noted that the Giemsa banding pattern of human chromosome 5 resembled that published by Stock and Hsu TM for an African green monkey small subtelocentric chromosome, which had been implicated in monkey interferon synthesis several years earlier by Cassingena e t al. a4 More recently, however, the two-chromosome requirement for human interferon production has been questioned. In human/Chinese hamster hybrids, apparently only the presence of human chromosome 5 is necessary for human interferon synthesis. TM We have concluded that both chromosomes 2 and 5 contain genes for human interferonl~; below we will briefly describe the interferon induction and assay procedures that we routinely use for studying interferon synthesis in somatic cell hybrids. For viral induction, we expose confluent monolayers of cells to Newcastle disease virus at 10 PFU/cell for 1 hr in 1 ml of serum-free medium. Cells are then washed with medium, and refed with fresh medium, containing 2% fetal bovine serum (FBS), for 24 hr. Medium is harvested and dialyzed against 0.15 M KCI pH 2 for several days at 4° to inactivate residual virus, and then against serum-free medium for 24 hr before assay. For simple poly(I), poly(C) inductions, 100 /~g of poly(I), poly(C) (P-L Biochemicals) per milliliter in phosphate-buffered saline (PBS) is added to confluent monolayers for 2 hr, after which the cells are washed and refed with medium containing 2% FBS. Medium is collected after 18-20 hr, centrifuged at low speed to remove any cell debris, and assayed. For poly(I) • poly(C) superinductions, 100/~g of poly(I) • poly(C) per milliliter are added in PBS to cells for 2 hr along with 20 /~g of cycloheximide (Sigma) and 50 ~g of DEAE-dextran (Pharmacia) per milliliter. After removal of the PBS and washing with serum-free medium, the cells are treated with cycloheximide (20 tzg/ml) for 3 hr in serum-free medium. The cycloheximide is then removed; the cell sheet is washed, and the cells are treated with 2 tzg of actinomycin D (Calbiochem) per milliliter for 1 hr. After washing, the cells are refed with medium containing 2% FBS; medium is collected after 18 hr for interferon assay. Medium incubated with hybrid cells after induction is assayed for interferon activity on both mouse (A9) and human (FS7 or GM 2504 Trisomy 21) fibroblasts. The assays are performed in 96-well microtiter trays (Linbro) by treating - 2 × 104 cells/well with various dilutions of la A. D. Stock and T. C. Hsu, Chromosoma 43, 211 (1973). 14 R. Cassingena, C. Chany, M. Vignal, H. Suarez, S. Estrade, and P. Lazar, Proc. Natl. Acad. Sci. U.S.A. 68, 580 (1971). ~5 M. J. Morgan and P. Faik, Br. J. Cancer 35, 254 (1977). ~ D. L. Slate and E H. Ruddle, Cell 16, 171 (1979).
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interferon preparations for 18-24 hr. The tray is vigorously inverted over paper toweling to remove residual interferon, and then 0.1 P F U of vesicular stomatitis virus per cell is added for 24 hr, at the end of which virusinduced cytopathic effects (CPE) are scored by examining the ceils under an inverted phase contrast microscope. Cell controls (no interferon, no virus), virus controls (no interferon, + virus), and interferon controls (treated with a known amount of human leukocyte or fibroblast interferon) are included in each assay. To determine the antigenic type of human interferon produced in a hybrid, antisera against human leukocyte and fibroblast interferons were obtained fi'om Dr. J. K. Dunnick (Antiviral Substances Program of the National Institute for Allergy and Infectious Diseases). They are titered against human interferon preparations of known specific activity in a modification of the CPE assay described above. Various antiserum dilutions (25/zl) are mixed with 25-/~1 samples containing 10 units of human interferon and incubated at 37° for 30 min. Human FS7 or GM 2504 cells are then added, and the assay is completed as usual. In neutralization tests for interferon produced by hybrid cells, approximately 10 units of human interferon are added to the dilution series of antibody preparations as above. In human/mouse hybrid cells containing human chromosomes 2 and 5, chromosome 5 can be eliminated by treatment with diphtheria toxin (0.25 Lf units/rrd, Connaught) for at least 48 hr. This selection procedure cannot be used with human/Chinese hamster hybrids, since the two species have approximately the same sensitivity to the toxin. Human/Chinese hamster hybrids tend not to retain human chromosome 2, and lines with 2-5 + karyotypes can be easily obtained. In our studies, we found that the presence of either human chromosome 2 or 5 could direct the synthesis of human interferon. With antibody neutralization tests performed on the interferon produced by hybrid cells, we determined that the human interferon was of the fibroblast (F) antigenic type. This was true when the human parental input was a fibroblast or when it was a peripheral blood leukocyte. The mouse or Chinese hamster parents of our hybrid cells were always fibroblast lines. The antibody neutralization data may therefore indicate that the epigenetic state of the parental rodent cell may influence the production of interferon of the segregating parental type. Meager et al. 1~ have reported that human chromosome 9 bears the gene for human fibroblast interferon. Human/mouse hybrid cells that retain an X/9 translocation chromosome produce human interferon, but lose this ,r A. Meager, H. G r a v e s , D. C. Burke, a n d D. M. Swallow, Nature (London) 2811, 493 (1978).
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ability when selected in 8-azaguanine. Meager e t al. 's interpretation of their data suggests that chromosomes 2 and 5 do not carry additional interferon loci, but some of their hybrids with 2 or 5 present (and 9 absent) produce low levels of human interferon. Since the hybrids we used for the mapping studies described above did not contain human chromosome 9, we attempted to confirm the results of Meager et al. lr by using a chromosome-mediated gene transfer line retaining only an X/9 translocation on a mouse A9 background (C. Miller and F. H. Ruddle, unpublished data). This line was generated by fusing isolated metaphase HeLa chromosomes to A9 cells. The procedure is outlined below (see also, Miller and RuddlelS). Approximately 10s mitotic cells are collected, pelleted gently (800 rpm, 5 min), and resuspended at 5 × 106 cells/ml in 0.075 M KCI, 0. I/xg of vinblastine per milliliter at room temperature for about 5 min. Cells are pelleted at 4°, resuspended in 15 mM "Iris • HCI, pH 7, 3 mM CaCI2 at 5 × 106 ceUs/ml, and put on ice for 15 min. Triton X-100 is added to 1%, and the cells are incubated at 37° for 10 min. Cells are then homogenized in a Dounce homogenizer (10-12 strokes) on ice and diluted with an equal volume of cold 15 mM Tris • HCI pH 7, 3 mM CaCI2. The suspension is then centrifuged at 800 rpm at 4° for I0 min, and the supernatant containing chromosomes is collected. (The pellet may be redissolved and centrifuged to recover any additional chromosomes.) Chromosomes are pelleted at 2000 rpm for approximately 30 min and then resuspended in HEPES buffer (NaC1, 8 g/liter; KCI, 0.37 g/liter; Na2HPO4 • 2 H20, 0.125 g/liter; dextrose, 1 g/liter; HEPES, pH 7.1, 5 g/liter) in about 30 ml per l0 s donor cells. An aliquot for chromosome counts is removed, and the rest is pelleted. The chromosomes should be resuspended at 2 × 10S/ml, with the suspension done in 7 : 1 HEPES buffer : 2 M CaCIz. Half the required amount of HEPES buffer is added; and the chromosomes are vigorously resuspended by vortex mixing, then the rest of the HEPES is added. Then the 2 M CaC12 is added, and the chromosomes are left at room temperature for about 30 min. Recipient cells should have been plated the previous day to be approximately 25% confluent when chromosomes are added. The medium is removed from the recipient ceils, and 2 ml of chromosome suspension are added per 75-cm z culture flask. Cells are incubated 30 min at room temperature. Next 20 ml of medium with fetal bovine serum and antibiotics are added to the flask and incubated for 4 hr at 37°. Dimethyl sulfoxide (DMSO) is added to a final concentration of 10%, and the cells are incubated for 30 min at room temperature. The DMSO medium is removed and replaced with fresh medium; the cells are incubated at 37° , and selective medium is added after 24-48 hr. la C. L. Miller and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 75, 3346 (1978).
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Human HeLa c e l l chromosomes + mouse A9 c e l l s (HPRT-) Chromosome mediated gene t r a n s f e r l l n e ~ t t h human (X,9) t r a n s l o c a t l o n
li!
Human/mouse hybrid with human chromosomes 2 and 5 (HPRT-)
Prolonged ~ t t o t i c a r r e s t C e n t r l f u ~ t l o n in c y t o c h a l a s / n Separation of u a 1 1 alcroce118
0 F~tcrocells
H1croce11 hybrids [2 +, 5+ , (X/9) +] FIG. 1. Generation of microcell hybrids containing human chromosomes 2 and 5 and an (X/9) translocation.
The chromosome-mediated gene transfer line retaining only the human X/9 translocation produced human interferon under virus induction and poly(I) • poly(C) superinduction. 19 In order to study the effect of the X/9 translocation chromosome on the other interferon loci, we transferred this chromosome to a human/mouse 2+5+ hybrid via the microcell procedure published by Fournier and Ruddle. ~° Interferon production in the resultant microcell hybrids is still being evaluated (Fig. 1). It has not been possible to determine whether the three human interferon loci described thus far are identical. Antigenically the interferons made by cells with various combinations of the three implicated chromosomes is fibroblast (F) type, but there may be differences in the molecules coded by each chromosome. It is highly unlikely that genes with exactly la D. L. Slate and F. H. Ruddle, A n n . N . Y. A c a d . Sci. 350, 174 (1980). 20 R. E. K. Fournier and F. H. Ruddle, Proc. Natl. Acad. Sci. U . S . A . 74, 319 (1977),
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the same nucleotide sequence exist on the three chromosomes implicated in human interferon synthesis. It is possible that regulatory genes and/or the epigenetic state of a cell may determine which gene(s) to transcribe in response to a given stimulus. Use of Aneuploid or Aneusomic Human Cells in Gene Mapping Another method for studying the genetic control of human interferon synthesis employs aneuploid or aneusomic human cell lines and depends on observing differences in interferon production depending on the dosage of specific chromosomes or portions of chromosomes. Tan and his colleagues 21 have reported studies with human fibroblasts with different numbers of copies of chromosome 5, some with more short arms than long arms of this chromosome, and others with this situation reversed. Their data suggest that the amount of human interferon produced was inversely related to the number of chromosome 5 short arms, and proportional to the number of chromosome 5 long arms. Slate and Ruddle 16 found that cri-du-chat fibroblasts, with deletions of part of the short arm of chromosome 5, produce less interferon than other human fibroblasts. It is unclear whether these results with aneuploid cell lines reflect true gene dosage relationships or merely represent the results of aberrant regulation in cells lacking a balanced set of genes required for the battery of steps involved in what is termed interferon induction. Interpretation of data obtained from established cell lines with rearranged karyotypes is very difficult when the parameter to be measured (interferon yield) varies over a wide range owing to a combination of genetic, epigenetic, and cell culture conditions. Mapping Other Human Interferon Genes As mentioned earlier, most of the somatic cell genetic studies on human interferon synthesis have been performed with human (aneuploid) fibroblasts or with rodent fibroblasts as the nonsegregating parent in human/rodent hybrids. These factors may be responsible for the fact that the only gene assignments thus far have been for human fibroblast interferon. Construction o f leukocyte × leukocyte hybrids or hybrids with the nonsegregating parent lacing a tumor cell derived from the immune system may allow the mapping of leukocyte and immune interferons. It will be extremely interesting to see whether these interferons are coded by the same chromosomes involved in fibroblast interferon synthesis. It may also 21 y. H, Tan, in "Interfcrons and Their Actions" (W. E. Stewart, II, ed.), pp. 73-90. CRC Press, Cleveland, Ohio, 1977.
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be possible to exploit the finding that small amounts of leukocyte interferon can be made by human fibroblasts induced with viruses; using suitable purification schemes or antigenic methods, this small amount of leukocyte interferon could be detected after induction offibroblast-derived cell hybrids and associated with the presence of a particular human chromosome. It is clear that we have only just begun to decipher the genetics behind human interferon synthesis. With cloned interferon gene probes, it will be possible to confirm or refute the current gene assignments and to learn in molecular detail about the organization of the human interferon genes. Acknowledgments We would like to thank Carol Miller for her gene transfer cell line. This work was supported by NIH Grant GM 09966and a grant from the American Cancer Societyto F.H.R.
[66] S o m a t i c C e l l G e n e t i c M e t h o d s for S t u d y o f S e n s i t i v i t y to Human Interferon
By DORIS L. SLATE and FRANK H. RUDDLE The genes governing production of interferon and sensitivity to its action have proved to be asyntenic in the species that have been studied) Somatic cell genetic analyses of interspecific heterokaryons and hybrids have indicated that interferon production and response genes from two different species can function in a single cell. If the two parental species produce interferons that do not show significant cross-species activity, then it is possible to map genes in interspecific hybrids selectively segregating the chromosomes of one input species. Chromosome assignments for genes governing sensitivity to interferon have been made in man 2,s and mouse (this volume [67]). In this chapter we will concentrate on the mapping of genes involved in the antiviral response to human interferon and discuss the recent somatic cell genetic studies that have attempted to determine the nature of the gene product(s) responsible for conferring interferon sensitivity to a cell. D. L. Slate a n d F. H. Ruddle, Pharmacol. Ther. 4, 221 (1979). Y. H. Tan, J. A. Tischfield, a n d F. H. Ruddle, J. Exp. Mecl. 137, 317 (1973). 3 C. C h a n y , M. Vignal, P. Couillin, N. V. Cong, J. Boue, and A. Bou¢, Proc. Natl. Acad, Sci. U.S.A. 72, 3129 (197S).
METHODS IN ENZYMOLOGY, VOL. 79
Copyright © 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979~5
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[65] M e t h o d s for M a p p i n g H u m a n I n t e r f e r o n Structural Genes By DORIS L. SLATE and FRANK H. RUDDLE
Studies on the human interferon system have revealed a complex set of genetic and epigenetic factors that influence the synthesis and action of interferon. Interferon induction requires de novo RNA and protein synthesis as determined through the use of transcription and translation inhibitors. 1 Cells that are actively synthesizing interferon can be enucleated, and the resulting cytoplasts can still produce interferon. 2 The yield of interferon can vary over a wide range for human cells, depending on such factors as the " a g e " of a culture, the inducing stimulus, and whether the cells have a limited life-span or are "transformed" to indefinite growth in culture. Not only do the cell type and induction regimen affect the amount of interferon made, they can also affect the type of interferon made. Three major types of human interferon have been described: fibroblast (F), leukocyte (Le), and immune (sometimes called T or type II) interferons. F type is usually produced in fibroblast cultures treated with poly(I) • poly(C); Le type is usually made in buffy coat cells or in virus-transformed established lymphoblastoid lines after viral induction. Immune interferon is produced by various cells of the immune system in response to several types of stimulation (for example, treatment with mitogens or exposure to antigens that trigger immune recognition). These human interferon species differ in a number of respects, including stability at pH 2 and high temperature, antigenically, in molecular weight, and in cross-species antiviral activity. Havell, Hayes, and Vil~ek~ have reported that human fibroblasts can synthesize both F and Le type interferons after viral induction. The human interferon system is also amenable to various treatments that can modulate the amount or time course of interferon synthesis. Two major examples are priming and superinduction. In priming, low doses of interferon are used to pretreat cells, which are then normally induced; interferon is produced earlier than in unprimed control cells treated with just the inducer. 4 Superinduction involves the use of suitably timed doses of translation and transcription inhibitors and induction with poly(I) 1 M. Ho and J. A. Armstrong, Annu, Rev. Microbiol. 29, 131 (1975). 2 D. C. Burke and G. Veomett, Proc. Natl. Acad. Sci. U.S.A. 74, 3391 (1977). 3 E. A. Havell, T. G. Hayes, and J. Vil~ek, Virology 89, 330 (1978). 4 W. E. Stewart, II, L. B. Gosser, and R. Z. Lockart, J. Virol. 7, 792 (1971).
METHODS IN ENZYMOLOGY, VOL, 79
Copyright ~ 1981 by Academic Press. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979-5
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poly(C). This technique lengthens the period of interferon synthesis and increases final yields. On a molecular level, superinduction increases the amount of interferon messenger RNA synthesized and also stabilizes this messenger.5,6 The genetic control of human interferon synthesis and mechanism of action has been examined in tissue culture systems via somatic cell hybridization and the study of aneuploid or aneusomic human cell lines. We will attempt to summarize the major techniques and findings in the mapping of genes governing human interferon production. Somatic Cell Hybrids in Human Interferon Gene Mapping Most of the somatic cell genetic studies that have resulted in human gene assignments have been performed with human/rodent hybrid cells. Human/mouse and human/Chinese hamster hybrid cell lines tend to lose human chromosomes preferentially, making it possible to correlate the presence or the absence of a specific human phenotype with the presence or the absence of a particular human chromosome. We will not describe the generation of hybrids, or the isozyme and karyotype analysis, required for gene mapping. Detailed procedures for these techniques can be found in the literature (for example, see Kennett7 for a discussion of hybridization methods, Nichols and Ruddle s and Kozak et al. 9 for starch gel electrophoretic conditions to distinguish mouse and human isozymes, and Kozak, Lawrence, and Ruddle 1° and Worton and Duff11 for karyotype procedures for analyzing hybrid cells). The first assignment for human genes involved in the synthesis of interferon was made by Tan, Creagan, and Ruddle. 12They characterized a large number of human/mouse hybrid lines and found that the ability to produce human interferon could not be associated with the presence of any one human chromosome. When they examined the correlation between human interferon production and the presence of two human chromosomes together, however, they found that when a hybrid con5 p. B. Sehgal and I. Tamm, Virology 92, 240 (1979). 6 R. L. Cavalieri, E. A. Havell, J. Vil~ek, and S. Pestka, Prec. Natl. Acad. Sci. U.S.A. 74, 4415 (1977). R. H. Kennett, this series, Vol. 58, p. 345. s E. A. Nichols and F. H. Ruddle, J. Histochern. Cytochem. 21, 1066 (1973). 9 C. A. Kozak, R. E. K. Fournier, L. A. Leinwand, and F. H. Ruddle, Biochem. Genet. 17, 23 (1979). ao C. A. Kozak, J. B. Lawrence, and F. H. Ruddle, Exp. Cell Res. 105, 109 (1977). 1~ R. G. Worton and C. Duff, this series, Vol. 58, p. 322. Y. H. Tan, R. P. Creagan, and F. H. Ruddle, Prec. Natl. Acad. Sci. U.S.A. 71, 2251 (1974).
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tained both human chromosomes 2 and 5, human interferon synthesis could be induced with proper stimulation. They also noted that the Giemsa banding pattern of human chromosome 5 resembled that published by Stock and Hsu TM for an African green monkey small subtelocentric chromosome, which had been implicated in monkey interferon synthesis several years earlier by Cassingena e t al. a4 More recently, however, the two-chromosome requirement for human interferon production has been questioned. In human/Chinese hamster hybrids, apparently only the presence of human chromosome 5 is necessary for human interferon synthesis. TM We have concluded that both chromosomes 2 and 5 contain genes for human interferonl~; below we will briefly describe the interferon induction and assay procedures that we routinely use for studying interferon synthesis in somatic cell hybrids. For viral induction, we expose confluent monolayers of cells to Newcastle disease virus at 10 PFU/cell for 1 hr in 1 ml of serum-free medium. Cells are then washed with medium, and refed with fresh medium, containing 2% fetal bovine serum (FBS), for 24 hr. Medium is harvested and dialyzed against 0.15 M KCI pH 2 for several days at 4° to inactivate residual virus, and then against serum-free medium for 24 hr before assay. For simple poly(I), poly(C) inductions, 100 /~g of poly(I), poly(C) (P-L Biochemicals) per milliliter in phosphate-buffered saline (PBS) is added to confluent monolayers for 2 hr, after which the cells are washed and refed with medium containing 2% FBS. Medium is collected after 18-20 hr, centrifuged at low speed to remove any cell debris, and assayed. For poly(I) • poly(C) superinductions, 100/~g of poly(I) • poly(C) per milliliter are added in PBS to cells for 2 hr along with 20 /~g of cycloheximide (Sigma) and 50 ~g of DEAE-dextran (Pharmacia) per milliliter. After removal of the PBS and washing with serum-free medium, the cells are treated with cycloheximide (20 tzg/ml) for 3 hr in serum-free medium. The cycloheximide is then removed; the cell sheet is washed, and the cells are treated with 2 tzg of actinomycin D (Calbiochem) per milliliter for 1 hr. After washing, the cells are refed with medium containing 2% FBS; medium is collected after 18 hr for interferon assay. Medium incubated with hybrid cells after induction is assayed for interferon activity on both mouse (A9) and human (FS7 or GM 2504 Trisomy 21) fibroblasts. The assays are performed in 96-well microtiter trays (Linbro) by treating - 2 × 104 cells/well with various dilutions of la A. D. Stock and T. C. Hsu, Chromosoma 43, 211 (1973). 14 R. Cassingena, C. Chany, M. Vignal, H. Suarez, S. Estrade, and P. Lazar, Proc. Natl. Acad. Sci. U.S.A. 68, 580 (1971). ~5 M. J. Morgan and P. Faik, Br. J. Cancer 35, 254 (1977). ~ D. L. Slate and E H. Ruddle, Cell 16, 171 (1979).
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interferon preparations for 18-24 hr. The tray is vigorously inverted over paper toweling to remove residual interferon, and then 0.1 P F U of vesicular stomatitis virus per cell is added for 24 hr, at the end of which virusinduced cytopathic effects (CPE) are scored by examining the ceils under an inverted phase contrast microscope. Cell controls (no interferon, no virus), virus controls (no interferon, + virus), and interferon controls (treated with a known amount of human leukocyte or fibroblast interferon) are included in each assay. To determine the antigenic type of human interferon produced in a hybrid, antisera against human leukocyte and fibroblast interferons were obtained fi'om Dr. J. K. Dunnick (Antiviral Substances Program of the National Institute for Allergy and Infectious Diseases). They are titered against human interferon preparations of known specific activity in a modification of the CPE assay described above. Various antiserum dilutions (25/zl) are mixed with 25-/~1 samples containing 10 units of human interferon and incubated at 37° for 30 min. Human FS7 or GM 2504 cells are then added, and the assay is completed as usual. In neutralization tests for interferon produced by hybrid cells, approximately 10 units of human interferon are added to the dilution series of antibody preparations as above. In human/mouse hybrid cells containing human chromosomes 2 and 5, chromosome 5 can be eliminated by treatment with diphtheria toxin (0.25 Lf units/rrd, Connaught) for at least 48 hr. This selection procedure cannot be used with human/Chinese hamster hybrids, since the two species have approximately the same sensitivity to the toxin. Human/Chinese hamster hybrids tend not to retain human chromosome 2, and lines with 2-5 + karyotypes can be easily obtained. In our studies, we found that the presence of either human chromosome 2 or 5 could direct the synthesis of human interferon. With antibody neutralization tests performed on the interferon produced by hybrid cells, we determined that the human interferon was of the fibroblast (F) antigenic type. This was true when the human parental input was a fibroblast or when it was a peripheral blood leukocyte. The mouse or Chinese hamster parents of our hybrid cells were always fibroblast lines. The antibody neutralization data may therefore indicate that the epigenetic state of the parental rodent cell may influence the production of interferon of the segregating parental type. Meager et al. 1~ have reported that human chromosome 9 bears the gene for human fibroblast interferon. Human/mouse hybrid cells that retain an X/9 translocation chromosome produce human interferon, but lose this ,r A. Meager, H. G r a v e s , D. C. Burke, a n d D. M. Swallow, Nature (London) 2811, 493 (1978).
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ability when selected in 8-azaguanine. Meager e t al. 's interpretation of their data suggests that chromosomes 2 and 5 do not carry additional interferon loci, but some of their hybrids with 2 or 5 present (and 9 absent) produce low levels of human interferon. Since the hybrids we used for the mapping studies described above did not contain human chromosome 9, we attempted to confirm the results of Meager et al. lr by using a chromosome-mediated gene transfer line retaining only an X/9 translocation on a mouse A9 background (C. Miller and F. H. Ruddle, unpublished data). This line was generated by fusing isolated metaphase HeLa chromosomes to A9 cells. The procedure is outlined below (see also, Miller and RuddlelS). Approximately 10s mitotic cells are collected, pelleted gently (800 rpm, 5 min), and resuspended at 5 × 106 cells/ml in 0.075 M KCI, 0. I/xg of vinblastine per milliliter at room temperature for about 5 min. Cells are pelleted at 4°, resuspended in 15 mM "Iris • HCI, pH 7, 3 mM CaCI2 at 5 × 106 ceUs/ml, and put on ice for 15 min. Triton X-100 is added to 1%, and the cells are incubated at 37° for 10 min. Cells are then homogenized in a Dounce homogenizer (10-12 strokes) on ice and diluted with an equal volume of cold 15 mM Tris • HCI pH 7, 3 mM CaCI2. The suspension is then centrifuged at 800 rpm at 4° for I0 min, and the supernatant containing chromosomes is collected. (The pellet may be redissolved and centrifuged to recover any additional chromosomes.) Chromosomes are pelleted at 2000 rpm for approximately 30 min and then resuspended in HEPES buffer (NaC1, 8 g/liter; KCI, 0.37 g/liter; Na2HPO4 • 2 H20, 0.125 g/liter; dextrose, 1 g/liter; HEPES, pH 7.1, 5 g/liter) in about 30 ml per l0 s donor cells. An aliquot for chromosome counts is removed, and the rest is pelleted. The chromosomes should be resuspended at 2 × 10S/ml, with the suspension done in 7 : 1 HEPES buffer : 2 M CaCIz. Half the required amount of HEPES buffer is added; and the chromosomes are vigorously resuspended by vortex mixing, then the rest of the HEPES is added. Then the 2 M CaC12 is added, and the chromosomes are left at room temperature for about 30 min. Recipient cells should have been plated the previous day to be approximately 25% confluent when chromosomes are added. The medium is removed from the recipient ceils, and 2 ml of chromosome suspension are added per 75-cm z culture flask. Cells are incubated 30 min at room temperature. Next 20 ml of medium with fetal bovine serum and antibiotics are added to the flask and incubated for 4 hr at 37°. Dimethyl sulfoxide (DMSO) is added to a final concentration of 10%, and the cells are incubated for 30 min at room temperature. The DMSO medium is removed and replaced with fresh medium; the cells are incubated at 37° , and selective medium is added after 24-48 hr. la C. L. Miller and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 75, 3346 (1978).
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Human HeLa c e l l chromosomes + mouse A9 c e l l s (HPRT-) Chromosome mediated gene t r a n s f e r l l n e ~ t t h human (X,9) t r a n s l o c a t l o n
li!
Human/mouse hybrid with human chromosomes 2 and 5 (HPRT-)
Prolonged ~ t t o t i c a r r e s t C e n t r l f u ~ t l o n in c y t o c h a l a s / n Separation of u a 1 1 alcroce118
0 F~tcrocells
H1croce11 hybrids [2 +, 5+ , (X/9) +] FIG. 1. Generation of microcell hybrids containing human chromosomes 2 and 5 and an (X/9) translocation.
The chromosome-mediated gene transfer line retaining only the human X/9 translocation produced human interferon under virus induction and poly(I) • poly(C) superinduction. 19 In order to study the effect of the X/9 translocation chromosome on the other interferon loci, we transferred this chromosome to a human/mouse 2+5+ hybrid via the microcell procedure published by Fournier and Ruddle. ~° Interferon production in the resultant microcell hybrids is still being evaluated (Fig. 1). It has not been possible to determine whether the three human interferon loci described thus far are identical. Antigenically the interferons made by cells with various combinations of the three implicated chromosomes is fibroblast (F) type, but there may be differences in the molecules coded by each chromosome. It is highly unlikely that genes with exactly la D. L. Slate and F. H. Ruddle, A n n . N . Y. A c a d . Sci. 350, 174 (1980). 20 R. E. K. Fournier and F. H. Ruddle, Proc. Natl. Acad. Sci. U . S . A . 74, 319 (1977),
[68l
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the same nucleotide sequence exist on the three chromosomes implicated in human interferon synthesis. It is possible that regulatory genes and/or the epigenetic state of a cell may determine which gene(s) to transcribe in response to a given stimulus. Use of Aneuploid or Aneusomic Human Cells in Gene Mapping Another method for studying the genetic control of human interferon synthesis employs aneuploid or aneusomic human cell lines and depends on observing differences in interferon production depending on the dosage of specific chromosomes or portions of chromosomes. Tan and his colleagues 21 have reported studies with human fibroblasts with different numbers of copies of chromosome 5, some with more short arms than long arms of this chromosome, and others with this situation reversed. Their data suggest that the amount of human interferon produced was inversely related to the number of chromosome 5 short arms, and proportional to the number of chromosome 5 long arms. Slate and Ruddle 16 found that cri-du-chat fibroblasts, with deletions of part of the short arm of chromosome 5, produce less interferon than other human fibroblasts. It is unclear whether these results with aneuploid cell lines reflect true gene dosage relationships or merely represent the results of aberrant regulation in cells lacking a balanced set of genes required for the battery of steps involved in what is termed interferon induction. Interpretation of data obtained from established cell lines with rearranged karyotypes is very difficult when the parameter to be measured (interferon yield) varies over a wide range owing to a combination of genetic, epigenetic, and cell culture conditions. Mapping Other Human Interferon Genes As mentioned earlier, most of the somatic cell genetic studies on human interferon synthesis have been performed with human (aneuploid) fibroblasts or with rodent fibroblasts as the nonsegregating parent in human/rodent hybrids. These factors may be responsible for the fact that the only gene assignments thus far have been for human fibroblast interferon. Construction o f leukocyte × leukocyte hybrids or hybrids with the nonsegregating parent lacing a tumor cell derived from the immune system may allow the mapping of leukocyte and immune interferons. It will be extremely interesting to see whether these interferons are coded by the same chromosomes involved in fibroblast interferon synthesis. It may also 21 y. H, Tan, in "Interfcrons and Their Actions" (W. E. Stewart, II, ed.), pp. 73-90. CRC Press, Cleveland, Ohio, 1977.
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be possible to exploit the finding that small amounts of leukocyte interferon can be made by human fibroblasts induced with viruses; using suitable purification schemes or antigenic methods, this small amount of leukocyte interferon could be detected after induction offibroblast-derived cell hybrids and associated with the presence of a particular human chromosome. It is clear that we have only just begun to decipher the genetics behind human interferon synthesis. With cloned interferon gene probes, it will be possible to confirm or refute the current gene assignments and to learn in molecular detail about the organization of the human interferon genes. Acknowledgments We would like to thank Carol Miller for her gene transfer cell line. This work was supported by NIH Grant GM 09966and a grant from the American Cancer Societyto F.H.R.
[66] S o m a t i c C e l l G e n e t i c M e t h o d s for S t u d y o f S e n s i t i v i t y to Human Interferon
By DORIS L. SLATE and FRANK H. RUDDLE The genes governing production of interferon and sensitivity to its action have proved to be asyntenic in the species that have been studied) Somatic cell genetic analyses of interspecific heterokaryons and hybrids have indicated that interferon production and response genes from two different species can function in a single cell. If the two parental species produce interferons that do not show significant cross-species activity, then it is possible to map genes in interspecific hybrids selectively segregating the chromosomes of one input species. Chromosome assignments for genes governing sensitivity to interferon have been made in man 2,s and mouse (this volume [67]). In this chapter we will concentrate on the mapping of genes involved in the antiviral response to human interferon and discuss the recent somatic cell genetic studies that have attempted to determine the nature of the gene product(s) responsible for conferring interferon sensitivity to a cell. D. L. Slate a n d F. H. Ruddle, Pharmacol. Ther. 4, 221 (1979). Y. H. Tan, J. A. Tischfield, a n d F. H. Ruddle, J. Exp. Mecl. 137, 317 (1973). 3 C. C h a n y , M. Vignal, P. Couillin, N. V. Cong, J. Boue, and A. Bou¢, Proc. Natl. Acad, Sci. U.S.A. 72, 3129 (197S).
METHODS IN ENZYMOLOGY, VOL. 79
Copyright © 1981by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181979~5