Hybrid Mammalian Cells Assemble Hybrid Ribosomes

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Hybrid Mammalian Cells Assemble Hybrid RibosomesAuthor(s): Peter J. Wejksnora and Jonathan R. WarnerSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 76, No. 11 (Nov., 1979), pp. 5554-5558Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/70495 .Accessed: 07/05/2014 19:01Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp .JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact support@jstor.org. .National Academy of Sciences is collaborating with JSTOR to digitize, preserve and extend access toProceedings of the National Academy of Sciences of the United States of America.http://www.jstor.org This content downloaded from 169.229.32.136 on Wed, 7 May 2014 19:01:30 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/action/showPublisher?publisherCode=nashttp://www.jstor.org/stable/70495?origin=JSTOR-pdfhttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/page/info/about/policies/terms.jspProc. Natl. Acad. Sci. USA Vol. 76, No. 11, pp. 5554-5558, November 1979 Biochemistry Hybrid mammalian cells assemble hybrid ribosomes (ribosomal protein/ribosomal RNA/emetine resistance/RNA-protein interactions) PETER J. WEJKSNORA AND JONATHAN R. WARNER Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 Communicated by Robert Palese Perry, August 10, 1979 ABSTRACT Hybrid cell lines formed by fusion of mouse 3T3 cells and Chinese hamster ovary (CHO) cells resistant to emetine, which have an altered 40S ribosomal protein, are generally sensitive to emetine. From most hybrid lines it was possible to select sublines resistant to emetine. The ribosomal components of three lines were studied: A34, an emetine-sen- sitive hybrid; A34/R3, an emetine-resistant derivative of A34; and A72, an emetine-sensitive hybrid that did not give rise to emetine-resistant sublines. Genetic and biochemical evidence suggests that in A34 both the mouse emetine sensitivity gene and the hamster emetine resistance gene are active, whereas in A34/R3 only the hamster emetine resistance gene is active and in A72 only the mouse emetine sensitivity gene is active. The ribosomes of all three sublines contained both mouse and hamster RNA, predominantly mouse. However, the 60S subunits had roughly equal amounts of the three mouse and hamster proteins that could be distinguished by two-dimensional elec- trophoresis, suggesting the association of mouse RNA with hamster ribosomal proteins. The emetine-resistant and eme- tine-sensitive 40S subunits could be separated by sedimentation in 0.5 M KCI. Resistant subunits contained predominantly mouse RNA, presumably associated with the hamster protein conferring emetine resistance. We conclude that hybrid cells can form bybrid ribosomes and that the amounts of ribosomal RNA and ribosomal protein of each species are not closely coupled. The biosynthesis of eukaryotic ribosomes is a complex process requiring the coordinated production of several RNA species and 70 different proteins as well as their assembly in the nucleolus (1). For mammalian cells, the mechanism by which such coordinated synthesis of protein and RNA is accomplished is little understood. Analysis of this system is hampered by the fact that ribosomes are essential for growth, making mutants difficult to obtain. Somatic cell hybrids provide an alternate route to examine the control of ribosome biosynthesis in mammalian cells. With respect to the synthesis of ribosomal RNA, hybrid cells that lose preferentially the chromosomes of one parent generally express only one type of 28S rRNA (2-4), whereas hybrids of two rodent species express both (5, 6). Recently it has been suggested that, in hybrids between Syrian hamster and mouse, individual cells contain active nucleolar organizer regions on chromosomes of both parents (7). However, in mouse-human hybrids only one parental nucleolar organizer is active, despite the presence of rDNA of both parents (4). Less attention has been paid to ri- bosomal proteins in hybrids. However, mouse and rat, and mouse and hamster, ribosomal proteins have been identified in hybrids between those species (8, 9). These studies did not distinguish between totipotent cells and subpopulations each of which produce the protein or RNA of a single species. As a practical matter, hybrid cells derived from the cross of two rodent species, which retain the chromosomes of both parents and maintain the ability to synthesize ribosomal com- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. ?1734 solely to indicate this fact. ponents of both species, are most amenable to study. This "coexistence" allows examination of ribosomal synthesis in cells in which failure to maintain the coordinated ribosome bio- synthesis of one parent is not lethal because a second functional set of genes for ribosomal components is also present. For such investigations to be fruitful the ribosomal components of each parent should be distinguishable, and ideally they should be selectable in vivo. Furthermore, the extent to which these components interact to form functional "hybrid" ribosomes must be determined because hybrid cells with separate and noninteracting systems for the biosynthesis of ribosomes should behave very differently from hybrids in which ribosomal components mix freely. In the study presented here, one parent is a Chinese hamster ovary (CHO) line requiring proline and resistant to the anti- biotic emetine (10). Mutants resistant to emetine have been shown to affect the 40S subunit (11), and one such mutant has an electrophoretically altered 40S protein (12). This protein has been tentatively identified as S14 (13), in the nomenclature of McConkey et al. (14). The complementary parent is mouse, whose chromosomes are readily distinguishable from those of CHO (15, 16), and whose genetics have been extensively studied (17, 18). We have established methods to detect differences in the ribosomal RNA and protein components of the two parents. In hybrids formed between CHO and mouse we have determined that the ribosomal components do interact to form hybrid ri- bosomes. Although roughly equal amounts of ribosomal proteins of both species are found, the ribosomal RNA is predominantly mouse. METHODS Cell Lines and Culture Techniques. A hamster CHO line requiring proline and resistant to emetine (emtR141), obtained from L. Siminovitch (10), and a mouse 3T3 thymidine kinase- negative line (C2F), obtained from C. Basilico (5), were rou- tinely grown at 370C as monolayers in 75-cm2 flasks (Corning) and fed with Dulbecco's modified Eagle's medium (DME) supplemented with proline at 40 mg/liter and 10% fetal calf serum (North American Biologicals, Miami, FL). Separation of Ribosomal Subunits. Cells (1-4 X 107) were harvested, washed two times with Earle's salt solution (19), suspended in 10 ml of 50 mM Tris acetate, pH 7/50 mM NH4CI/12 mM MgCI2/1 mM dithiothreitol (TMN) for 15 min. Nonidet P-40 was added to 0.5% and the cells were homoge- nized with a Dounce homogenizer to ensure lysis. The nuclei were removed by centrifugation. The supernatant was layered over 10 ml of 10% sucrose in TMN and centrifuged 2 hr at 40,000 rpm in a Spinco Ti 60 rotor at 4?C. The pellet was Abbreviations: DME, Dulbecco's modified Eagle's medium; TMN, Tris acetate/NH4CI/MgCI2/dithiothreitol; KMT, KCI/MgCl2/Tris- HCI; NaDodSO4, sodium dodecyl sulfate; HAT, hypoxanthine/ami- nopterin/thymidine. 5554 This content downloaded from 169.229.32.136 on Wed, 7 May 2014 19:01:30 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jspBiochemistry: Wejksnora and Warner Proc. Natl. Acad. Sci. USA 76 (1979) 5555 washed quickly with 0.5 M KCI/5 mM MgCl2/20 mM Tris- HCI, pH 7.4 (KMT) buffer and resuspended in that buffer. Puromycin was added to 0.2 mM, and the suspension was in- cubated at 37?C for 25 min and centrifuged 10,000 X g for 10 min. The supernatant was layered over a 10-25% sucrose gra- dient in KMT buffer and centrifuged at 23,000 rpm for 23 hr in a Spinco SW 27 rotor at 4?C. The gradient was collected from the bottom and absorbance was monitored at 260 nm. Ribosomal Proteins. Proteins were extracted from sus- pended ribosomes by addition of MgCl2 to 0.1 M, dithiothreitol to 0.01 M, followed by 2 vol of glacial acetic acid (20). Samples were analyzed on a two-dimensional polyacrylamide gel modified (20) from that described by Mets and Bogorad (21). Ribosomal RNA. Ribosomal RNA was extracted with phenol from cytoplasmic fractions treated wth 1% sodium dodecyl sulfate (NaDodSO4) and resolved on sucrose gradients con- taining NaDodSO4 (22). To distinguish mouse from hamster RNA, 10 ,ug of 1 S or 28S RNA prepared from cells incubated 24 hr with [32P]phosphate in supplemented DME was mixed with 100 ,ug of unlabeled RNA, precipitated with ethanol, air dried, and resuspended in 20 ,ul of 10 mM Tris-HCI, pH 7.4/1 mM EDTA. Fifteen units of ribonuclease Ti (Calbiochem) was added, the mixture was incubated 20 min at 370C, and the fragments were separated by two-dimensional polyacrylamide gel electrophoresis (23). The slab gel was wrapped in plastic wrap and exposed to x-ray film (Kodak NS-5) at -20?C for 1-3 days. To quantitate individual spots, the gel was laid on the developed film and the chosen spots were excised with a cork borer. A second film was exposed and developed to check the accuracy of the excision. The gel spots were digested in 0.5 ml of 30% H202 at 600C, mixed with 10 ml of Aquasol (New En- gland Nuclear), and assayed for radioactivity. Cell Hybridization and Selection. Techniques utilized to fuse rodent cells and select hybrids were as described by Pon- tecorvo (24). Briefly, 7 X 105 of each cell type were plated to- gether on a 60-mm diameter dish (Falcon), grown overnight, washed repeatedly with polyethylene glycol (BDH 6000) in DME, and then incubated 24 hr in DME supplemented with proline and 10% fetal calf serum. The cells were then replated in 100-mm diameter dishes at concentrations of 1 X 104 or 5 X 105 cells per dish in DME with 100 ,uM hypoxanthine/0.4 jiM aminopterin/16 jiM thymidine (HAT) and 10% dialyzed fetal calf serum but without proline. Appropriate controls for re- version of both parental markers were treated similarly. Colonies that arose from the hybrid cross were picked with cloning rings and cultured in the same medium. Clones were prepared by seeding cells on glass fragments and picking fragments with only one cell. Metaphase chromosomes were prepared as described by Kozak et al. (16), enabling us to dif- ferentiate the small telocentric mouse chromosomes from hamster chromosomes and thus to verify the hybrid nature and homogeneity of the clone. To determine emetine resistance, 103-105 cells were seeded in 100-mm dishes containing DME with HAT and dialyzed fetal calf serum with or without eme- tine at 0.1 ,uM and examined after 5-14 days. At this concen- tration of emetine the plating efficiency of emetine-resistant CHO cells is 50%, while that of 3T3 cells is less than 1o-5. We designated as sensitive the hybrids with a plating efficiency of less than 1%. To select emetine-resistant clones from sensitive hybrids, 5 X 106 cells were plated at 5 X 105 cells per 100-mm dish in the presence of 0.1 ,uM emetine. After 2 weeks colonies that arose were picked and cultured several days in the presence of 0.1 ,uM emetine. Emetine-resistant cells were then routinely grown in the same media as other hybrids. During prolonged growth, emetine resistance was occasionally verified. RESULTS Ribosomal RNA and Proteins of Mouse and Hamster Cells. Ribosomal proteins from CHO and 3T3 were separated by two-dimensional electrophoresis at pH 5 in the first dimension, with NaDodSO4 in the second. As shown by using another gel system (9), the patterns (Fig. 1 A and B) are similar but not identical. At least three proteins of the mouse, all from the 60S subunit (Fig. 1A), are resolved from their hamster counterparts (Fig. 1B) under these conditions. These differences are seen most clearly in the pattern of proteins from the hybrid line A34 (Fig. 1C), which contains both parental types. The proteins in the upper pair of spots, which are probably L6 (13, 14), differ slightly in molecular weight. Those in the middle pair appear to differ in size by several thousand daltons, on the basis of molecular weight standards in the second-dimensional gel. The proteins in the lower pair, which stain rather faintly but are clear on the original gel, differ in charge but not in size. The basic identity of the proteins in each pair has been verified by proteolytic digestion of the spots followed by a third dimension of electrophoresis, as in the method of Cleveland et al. (25). S14, the protein thought to be responsible for emetine resistance (12), A B C * r a _e s ~~: ge cre a~~~~~~~ FIG. 1. Ribosomal proteins of mouse, hamster, and hybrid. The 805 ribosomes were prepared from cultures containing 1 X i07 cells, and proteins were extracted by 67% acetic acid as described (4). Samples containing approximately 100 ug of protein were lyophilized and separated by two-dimensional polyacrylamide gel electrophoresis (20). Separation from left to right was toward the cathode at pH 5, and separation from top to bottom was in the presence of NaDodSO4. (A) Mouse; (B) hamster. Unique mouse proteins are marked by solid arrows, their hamster counterparts by dashed arrows. The protein shown to be altered in other emetine-resistant CHO lines is marked with an asterisk (12). (C) Proteins extracted from the emetine-sensitive hybrid A34. Mouse proteins are indicated by solid arrows, hamster proteins by dashed arrows. This content downloaded from 169.229.32.136 on Wed, 7 May 2014 19:01:30 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jsp5556 Biochemistry: Wejksnora and Warner Proc. Natl. Acad. Sci. USA 76(1979) A BC -t_~~~~~P 40~_ D E F FIG. 2. Ribosomal RNA of mouse, hamster, and hybrid cells digested with ribonuclease Ti. Cells of each type (2 X 106) were incubated for 2 hr with 1 mCi (1 Ci = 3.7 X 101? becquerels) of [32P]phosphate in DME with reduced phosphate concentration and then for 12 hr in complete DME. RNA was prepared in NaDodSO4/phenol from mouse (A, D) or hamster (B, E) cytoplasm or separated hybrid ribosomal subunits (C, F) (22). The 28S and 18S species were separated on sucrose gradients, precipitated from 67% EtOH, digested with 15 units of Ti ribonuclease for 20 min at 37?C, separated by two-dimensional polyacrylamide gel electrophoresis (23), and autoradiographed. Mouse RNA is shown in A (28S) and D (18S), with solid arrows indicating unique mouse spots; hamster RNA is shown in B (28S) and E (18S), with dashed arrows indicating unique spots. RNA obtained from hybrid A34/R3 60S subunits (C) and degraded 40S subunit (F) (see peak Y of Fig. 5B) shows spots unique to both mouse (solid arrows) and hamster (dashed arrows). is indicated in Fig. lB by the asterisk. In the mutant we are studying, however, that protein is indistinguishable from wild-type hamster or mouse protein S14. One can distinguish RNA species of hamster and mouse cells by analysis of Ti digests of 32P-labeled RNA (Fig. 2). For the 18S species mouse-specific spots are clearly seen and in the 28S pattern spots unique both to mouse and to hamster are clearly resolved. When mouse and hamster RNA are mixed and then analyzed, the radioactivity present in these species-specific spots is an accurate measure of the amount of each parental type in the input. 40S Subunits of Emetine-Resistant Cells. An unexpected instability of emetine-resistant 40S subunits has permitted us to separate 40S subunits containing a hamster protein from those containing the mouse counterpart. This instability is observed after sedimentation through a sucrose gradient containing 0.5 A B 1.0 60S 40S 60S 40S X y - Sedimentation Fl(.. 3. Separation of ribosomal subunits from mouse (A) and emetine-resistant hamster (B) cells. Cells of each species (1 X 107) were swollen and lysed in TMN buffer. The nuclei were removed by centrif'ugation. Ribosomes were sedimented through 10% sucrose in that buf'f'er. The pellet was resuspended in KMT and incubated at 37?C for 20 min with 0.2 mM puromycin. This was layered over a 10-25% sucrose gradient in KMT buffer and centrifuged at 23,000 rpm in a Spinco SW 27 rotor for 23 hr at 4?C. M KCl (Fig. 3). A single 40S peak is obtained from mouse or wild-type CHO (not shown) under these conditions (Fig. 3A). However, two particles containing 18S RNA are obtained from emetine-resistant cells (Fig. SB). The heavier species (X) con- tains all but three of the normal 40S proteins (Fig. 4 A and B) and sediments about 10% slower than a normal 40S subunit. The lighter species (Y), which sediments well away from the 40S region, contains only about half the normal proteins (Fig. 4C). The loss of proteins is specific and nearly quantitative, suggesting their dissociation as discrete groups. The ribosomal protein associated with emetine resistance (12) is absent from both the X and Y species. The missing proteins can be detected at the top of the gradient. They are also found in intact 80S ri- bosomes (Fig. 1). Fusion and Selection. Mouse 3T3 and hamster CHO cells were fused and hybrids were selected by growth in HAT me- dium lacking proline. The efficiency of hybridization was 3 X 1o-3. Hybrid colonies were isolated and maintained in DME + HAT. Metaphase preparations were stained to detect mouse and hamster chromosomes. Chromosome counts were per- formed on each hybrid clone to verify both the hybrid nature of the cells and the homogeneity of the clone (Table 1). The hybrid cells were found to be sensitive to emetine (Table 1). We then attempted to isolate resistant subclones by growth in the presence of emetine. Resistant colonies were derived from hybrid A34 at a frequency of 3 X 10-6. One of these subclones. A34/R3, was examined and found to contain both mouse and hamster chromosomes (Table 1). Another hybrid line, A72, which had lost more than half of the hamster chromosomes, failed to produce any emetine-resistant colonies (Biochemistry: Wejksnora and Warner Proc. Natl. Acad. Sci. USA 76 (1979) 5557 A BeC, ci ~ ~ ~ ~ ~ c * ~ ~ ~ ~ ~ .* FIG. 4. Proteins from intact and degraded 40S subunits. Proteins were extracted from 40S ribosomal subunits prepared from mouse and from the X and Y peaks from emetine-resistant hamster cells and separated by two-dimensional gels. (A) Proteins from mouse. S14, the protein whose mutation can cause emetine resistance, is marked with an asterisk. (B) Proteins from the heavier (X) hamster peak. Proteins present in 40S subunits but missing here are indicated by dashed arrows. (C) Proteins from the lighter (Y) hamster peak. Proteins present in peak X, but missing in Y, are indicated by solid arrows. appeared that equal amounts of both parental species were present. To quantitate this observation, '4C-labeled ribosomal proteins from hybrid cells were mixed with unlabeled mouse and hamster ribosomal proteins and separated electrophoreti- cally. The specific spots were excised, the radioactivities were determined, and the ratios are shown in Table 2. The ratio for these proteins is nearly 1:1 in all three hybrids. 32P-Labeled ribosomal RNA was isolated from all three hy- brid lines. It was digested with TI, fragments were separated on two-dimensional gels, and the radioactivity present in species-specific spots was determined. The ratios of the two parental RNAs were calculated (Table 2). For hybrids A34 and A34/RS both parental types were clearly present, though mouse accounted for 80-90% of the total. For hybrid A72, mouse again predominated and hamster RNA, if present, was at the limit of resolution. Hybrid Cells Assemble Hybrid Ribosomes. Sucrose gra- dient profiles of the ribosomal subunits of two hybrid lines, A72 and A34, both emetine-sensitive, and the resistant subline A34/R3 are shown in Fig. 5. For A72, only the normal 40S subunit was seen (Fig. SC), suggesting that it contains only the mouse-derived emetine-sensitive protein. The pattern shown by hybrid A34 (Fig. 5A) is consistent with an equal mixture of sensitive and resistant subunits. This was demonstrated directly by acrylamide gel analysis of the proteins of the "40 + X" peak. On the other hand, the emetine-resistant subline, A34/R3 (Fig. Table 1. Characteristics of parental and hybrid cells Plating Emetine No. of efficiency sensitivity chromosomes* in 0.1 AM of 40S Cells Mode Mouse/CHO emetine, % subunitst Parents Mouse 69 + 4 5558 Biochemistry: Wejksnora and Warner Proc. Natl. Acad. Sci. USA 76 (1979) A B C 1.0 60S 40S 60S 40S 60S 0 /z1 1 x X y Sedimentation FIG. 5. Separation of ribosomal subunits from emetine-sensitive hybrids A34 (A) and A72 (C), and emetine-resistant hybrid A34/R3 (B), prepared and analyzed as in Fig. 3. in approximately equal amounts. However, the 28S RNA is predominantly mouse, indicating that some hamster 60S pro- teins must be present on subunits containing mouse RNA. This is particularly true for hybrid A72, which has virtually no hamster RNA but does contain hamster proteins. DISCUSSION The emetine-resistant protein causes the 40S subunit to be un- stable at high ionic strength, due to the loss of discrete groups of proteins. This occurs in 40S particles containing either hamster or mouse 18S RNA. Such instability was presaged by the finding that 40S subunits isolated from emetine-resistant cells had low activity for protein synthesis (11). The resistance to emetine, therefore, may arise from an alteration in the ar- chitecture of the 40S subunit, rather than from an alteration in binding to the mutated protein. As a practical matter, the in- stability and the concomitant ability to separate physically wild-type and emetine-resistant subunit is useful in the analysis of hybrid cells. Interspecific hybrids between emetine-sensitive mouse cells and emetine-resistant hamster cells are phenotypically eme- tine-sensitive. Similar results were obtained for intraspecific hybrids (26), in spite of the fact that in vitro experiments suggest the presence of some emtine-resistant ribosomes. Fig. 5A demonstrates directly that hybrid A34 contains both resistant and sensitive 40S subunits. Its sensitive phenotype therefore must be due to cooperativity between resistant and sensitive subunits, presumably as they traverse the same mRNA. It is possible, however, to select emetine-resistant hybrid lines that contain both mouse and hamster chromosomes. Because these arise at a level of about 3 X 10-5, and because no eme- tine-resistant cell of mouse origin has ever been isolated, we conclude that the mouse genes responsible for the protein conferring emetine sensitivity have been either lost or effi- ciently repressed. These cells appear to have few, if any, sen- sitive 40S subunits (Fig. 5B). Conversely, we were unable to isolate emetine-resistant clones from some hybrid lines-e.g., A72-suggesting that these cells have lost or repressed the gene responsible for the protein conferring emetine resistance. This suggestion is supported by the finding that these cells have few if any resistant 40S subunits (Fig. 5C). Ribosomes from hybrid cells contain both mouse and hamster RNA and both mouse and hamster proteins. However, because mouse RNA predominates (Fig. 2 C and F), whereas there are roughly equal amounts of those proteins we can differentiate (Fig. 1C), there is no strict coordination by species. On the contrary, it is clear for the 60S particle that hamster proteins must be assembled with mouse RNA. In fact, hamster 60S proteins are present in equal amounts even when hamster RNA is barely detectable, as in clone A72. It is testament to the con- servative evolution of ribosomal proteins that hamster proteins that differ significantly from their mouse counterparts are not discriminated against either by the nucleolus or by mouse ri- bosomal precursor RNA during the assembly of a 60S sub- unit. Direct evidence for the existence of hybrid ribosomes is also derived from analyzing the 40S particles of A34/R3. By the criteria of their emetine phenotype, and of their instability in 0.5 M KCI, these must contain the hamster-derived emetine- resistance protein (Table 1), yet the bulk of their RNA is mouse 18S. More interesting, perhaps, is the finding that in a hybrid cell there can be a complete divorce of the accumulation of the RNA of one species from the accumulation of the ribosomal proteins of that species. Further experiments are necessary to determine whether this occurs at the level of synthesis of the macromolecules or in the processing and assembly steps in- volved in the formation of the ribosome. We are grateful to Drs. L. Siminovitch, D. Roufa, and C. Basilico for cell lines and to Drs. P. Stanley and S. Skoultchi for advice on cell hybrids and for a critical reading of an early draft of this manuscript. This work was supported by American Cancer Society Grant NP 721, National Institutes of Health Grant P 30 CA 13330, and National In- stitutes of Health Grant 2 P50 GM 19100. 1. Warner, J. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. & Lengyel, P. (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 461-488. 2. Elicieri, G. L. & Green, H. (1969) J. Mol. Biol. 41, 252-260. 3. Bramwell, M. E. & Handmaker, S. D. (1971) Biochim. Biophys. Acta 232, 580-583. 4. Perry, R. P., Kelley, D. E., Schibler, U., Huebner K. & Croce, C. M. (1979) J. Cell. Physiol. 98,553-560. 5. Toniolo, D. & Basilico, C. (1974) Nature (London) 248, 411- 413. 6. Elicieri, G. L. (1972) J. Cell Biol. 53, 177-184. 7. Miller, 0. J., Dev. V. G., Miller, D. A., Tantravahi, R. & Elicieri, G. L. (1978) Exp. Cell Res. 115, 457-460. 8. Kuter, D. J. & Rodgers, A. (1975) Exp. Cell Res. 92,317-325. 9. Fujisawa, T. & Elicieri, G. L. (1975) Biochim. Biophys. Acta 402, 238-243. 10. Gupta, R. S. & Siminoviteh, L. (1976) Cell 9,213-219. 11. Gupta, R. S. & Siminovitch, L. (1977) Cell 10, 61-66. 12. Boersma, D., McGill, S., Mallenkamp, J. & Routa, D. S. (1979) J. Biol. Chem. 254, 559-567. 13. Madjar, J. J., Arpin, M., Buisson, M. & Reboud, J. P. (1979) Mol. Gen. Genet. 171, 121-134. 14. McConkey, E. H., Bielka, H., Gordon, J., Lastick, S. M., Lin, A., Ogata, K., Reboud, J.-P., Traugh, J. A., Traut, R. R., Warner, J. R., Welfle, H. & Wool, I. G. (1979) Mol. Gen. Genet. 169, 1-6. 15. Deaven, L. I. & Petersen, D. F. (1973) Chromosoma 41, 129- 144. 16. Kozak, C. A., Lawrence, J. B. & Ruddle, F. H. (1977) Exp. Cell Res. 105, 109-117. 17. Miller, D. A., Tantravahi, R., Dev, V. G. & Miller, 0. J. (1976) Genetics 84, 67-75. 18. Nesbitt, M. N. & Francke, U. (1973) Chromosoma 41, 145- 158. 19. Earle, W. R. (1943) J. Natl. Cancer Inst. 4, 165-212. 20. Warner, J. R. (1977) J. Mol. Biol. 115,315-333. 21. Mets, L. & Bogorad, L. (1974) Anal. Biochem. 57, 200-210. 22. Warner, J. R., Soeiro, R., Birnboim, H. C., Girard, H. & Darnell, J. E. (1966) J. Mol. Biol. 19,349-361. 23. Kennedy, S. I. T. (1976) J. Mol. Biol. 108, 491-511. 24. Pontecorvo, G. (1976) Somatic Cell Genet. 1, 397-400. 25. Cleveland, D. W., Fischer, S., Kirschner, M. & Ulrichk, K. (1977) J. Biol. Chem. 252, 1102-1106. 26. Gupta, R. S. & Siminovitch, L. (1978) Somatic Cell Genet. 4, 77-94. This content downloaded from 169.229.32.136 on Wed, 7 May 2014 19:01:30 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jspArticle Contentsp. 5554p. 5555p. 5556p. 5557p. 5558Issue Table of ContentsProceedings of the National Academy of Sciences of the United States of America, Vol. 76, No. 11 (Nov., 1979), pp. 5413-6022Growth of Complex Systems can be Related to the Properties of their Underlying Determinants [pp. 5413-5417]Sulfur Base Ligation to Iron (II) and Cobalt (II) Porphyrins [pp. 5418-5420]Significant Structure Theory Applied to Electrolyte Solution [pp. 5421-5423]Lower Bounds for Eigenvalues of Self-Adjoint Problems [pp. 5424-5425]Phosphorus in Connecticut Lakes Predicted by Land Use [pp. 5426-5429]RNA Ligase Reaction Products in Plasmolyzed Escherichia coli Cells Infected by T4 Bacteriophage [pp. 5430-5434]Molecular Cloning of Human $\epsilon $-Globin Gene [pp. 5435-5439]Implantation of the Isolated Human Erythrocyte Anion Channel into Plasma Membranes of Friend Erythroleukemic Cells by Use of Sendai Virus Envelopes [pp. 5440-5444]Rapid ATP Assays in Perfused Mouse Liver by $^{31}$P NMR [pp. 5445-5449]Binding of Inhibitor Alters Kinetic and Physical Properties of Extracellular Cyclic AMP Phosphodiesterase from Dictyostelium discoideum [pp. 5450-5454]Properties and Localization of $\beta $-endorphin Receptor in Rat Brain [pp. 5455-5459]Photoreactive Labeling of M13 Coat Protein in Model Membranes by Use of a Glycolipid Probe [pp. 5460-5464]Distinctive Nucleotide Sequences of Promoters Recognized by RNA Polymerase Containing a Phage-Coded ``$\sigma $-Like'' Protein [pp. 5465-5469]Altered Promoter Selection by a Novel form of Bacillus subtilis RNA Polymerase [pp. 5470-5474]Participation of Guanine Nucleotides in Nucleation and Elongation Steps of Microtubule Assembly [pp. 5475-5479]Initiation of Escherichia coli Ribosomal RNA Synthesis in vivo [pp. 5480-5484]Purification of Chicken Intestinal Receptor for 1,25-dihydroxyvitamin D [pp. 5485-5489]Protein Synthesis in Rabbit Reticulocytes: Characteristics of a Postribosomal Supernatant Factor that Reverses Inhibition of Protein Synthesis in Heme-Deficient Lysates and Inhibition of Ternary Complex (Met-tRNA$_{\text{f}}{}^{\text{Met}}\cdot $eIF-2$\cdot $GTP) Formation by Heme-Regulated Inhibitor [pp. 5490-5494]Antibodies to Small Nuclear RNAs Complexed with Proteins are Produced by Patients with Systemic Lupus Erythematosus [pp. 5495-5499]Endonucleolytic Activity Directed towards 8-(2-hydroxy-2-propyl) Purines in Double-Stranded DNA [pp. 5500-5504]Myxobacterial Hemagglutinin: A Development-Specific Lectin of Myxococcus xanthus [pp. 5505-5509]Extracts of Drosophila Embryos Mediate Chromatin Assembly in vitro [pp. 5510-5514]Evidence for Hydrophobic Region within Heavy Chains of Mouse B Lymphocyte Membrane-Bound IgM [pp. 5515-5519]Regulation of Synthesis of a Major Outer Membrane Protein: Cyclic AMP Represses Escherichia coli Protein III Synthesis [pp. 5520-5523]Attenuation in the Escherichia coli Tryptophan Operon: Role of RNA Secondary Structure Involving the Tryptophan Codon Region [pp. 5524-5528]Control of Phosphoenolpyruvate-Dependent Phosphotransferase-Mediated Sugar Transport in Escherichia coli by Energization of the Cell Membrane [pp. 5529-5533]Complementation of the Temperature-Sensitive Defect in H5ts125 Adenovirus DNA Replication in vitro [pp. 5534-5538]Covalent Association of Protein with Replicative Form DNA of Parvovirus H-1 [pp. 5539-5543]Attractants and Repellents Control Demethylation of Methylated Chemotaxis Proteins in Escherichia coli [pp. 5544-5548]Vitamin D Metabolism during Pregnancy and Lactation in the Rat [pp. 5549-5553]Hybrid Mammalian Cells Assemble Hybrid Ribosomes [pp. 5554-5558]Ricin Linked to Monophosphopentamannose Binds to Fibroblast Lysosomal Hydrolase Receptors, Resulting in a Cell-Type-Specific Toxin [pp. 5559-5562]Termination of Transcription in Nucleoli Isolated from Tetrahymena [pp. 5563-5566]Spliced Adenovirus-Associated Virus RNA [pp. 5567-5571]Replication at Restrictive Temperatures in Escherichia coli Containing a polC$_{\text{ts}}$ Mutation [pp. 5572-5576]Solubilization of the Low Density Lipoprotein Receptor [pp. 5577-5581]Evidence for a Dissociable Protein Subunit Required for Calmodulin Stimulation of Brain Adenylate Cyclase [pp. 5582-5586]Inactive mRNA-Protein Complexes from Mouse Sarcoma-180 Ascites Cells [pp. 5587-5591]Biotinyl 5$^{\prime}$-adenylate: Corepressor Role in the Regulation of the Biotin Genes of Escherichia coli K-12 [pp. 5592-5595]Synthesis of Simian Virus 40 t Antigen in Escherichia coli [pp. 5596-5600]Purification and Partial Characterization of Human Lymphoblastoid Interferon [pp. 5601-5605]Glutathione: Interorgan Translocation, Turnover, and Metabolism [pp. 5606-5610]Differentiation as a Requirement for Simian Virus 40 Gene Expression in F-9 Embryonal Carcinoma Cells [pp. 5611-5615]Structural Studies of "Active Complex" of Bleomycin: Assignment of Ligands to the Ferrous ion in a Ferrous-bleomycin-Carbon Monoxide Complex [pp. 5616-5620]Glycoproteins of Sendai Virus are Transmembrane Proteins [pp. 5621-5625]Coupling of Opiate Receptors to Adenylate Cyclase: Requirement for Na$^{+}$ and GTP [pp. 5626-5630]Detection of Proteins like Human $\gamma $ and $\beta $ Globins in Escherichia coli Carrying Recombinant DNA Plasmids [pp. 5631-5635]Chromophore Organization in Photosynthetic Reaction Centers: High-Resolution Magnetophotoselection [pp. 5636-5640]Anisotropic Molecular Motion on Cell Surfaces [pp. 5641-5644]Lateral Mobility of an Amphipathic Apolipoprotein, ApoC-III, Bound to Phosphatidylcholine Bilayers with and without Cholesterol [pp. 5645-5649]Rotational Diffusion of Cell Surface Components by Time-Resolved Phosphorescence Anisotropy [pp. 5650-5654]Photoreversible Absorbance Changes in Solutions of Allophycocyanin Purified from Fremyella diplosiphon: Temperature Dependence and Quantum Efficiency [pp. 5655-5659]Globin Synthesis and Erythroid Differentiation in a Friend Cell Variant Deficient in Heme Synthesis [pp. 5660-5664]Growth of Cultured Human Epidermal Cells into Multiple Epithelia Suitable for Grafting [pp. 5665-5668]Amplified Dihydrofolate Reductase Genes in Unstably Methotrexate-Resistant Cells are Associated with Double Minute Chromosomes [pp. 5669-5673]An Endothelial Cell Growth Factor from Bovine Hypothalamus: Identification and Partial Characterization [pp. 5674-5678]Interaction of Physalaemin, Substance P, and Eledoisin with Specific Membrane Receptors on Pancreatic Acinar Cells [pp. 5679-5683]Introduction and Expression of a Rabbit $\beta $-globin Gene in Mouse Fibroblasts [pp. 5684-5688]Hormone Receptor Topology and Dynamics: Morphological Analysis Using Ferritin-Labeled Epidermal Growth Factor [pp. 5689-5693]Cellular Localization of the Insect Prothoracicotropic Hormone: In vitro Assay of a Single Neurosecretory Cell [pp. 5694-5698]Calcium-Dependent Increase in Adenosine 3$^{\prime}$,5$^{\prime}$-monophosphate and Induction of the Acrosome Reaction in Guinea Pig Spermatozoa [pp. 5699-5703]Interactions of Tumor Cells with Vascular Endothelial Cell Monolayers: A Model for Metastatic Invasion [pp. 5704-5708]Inhibition of Polyisoprenoid and Glycoprotein Biosynthesis Causes Abnormal Embryonic Development [pp. 5709-5713]Passage of Phenotypes of Chemically Transformed Cells Via Transfection of DNA and Chromatin [pp. 5714-5718]Relationship between Movement and Aggregation of Centrioles in Syncytia and Formation of Microtubule Bundles [pp. 5719-5723]In vitro Synthesis of a Putative Precursor of Mitochondrial Ornithine Transcarbamoylase [pp. 5724-5727]Estimation of Membrane Potentials of Individual Lymphocytes by Flow Cytometry [pp. 5728-5730]Epidermal Growth Factor Stimulation of DNA Synthesis is Potentiated by Compounds that Inhibit its Clustering in Coated Pits [pp. 5731-5735]Prevention of Genetic Anemias in Mice by Microinjection of Normal Hematopoietic Stem Cells into the Fetal Placenta [pp. 5736-5740]Phosphoethanolamine as a Growth Factor of a Mammary Carcinoma Cell Line of Rat [pp. 5741-5744]Purification of Cytoplasmic Tubulin and Microtubule Organizing Center Proteins Functioning in Microtubule Initiation from the Alga Polytomella [pp. 5745-5749]Early Termination of Heterogeneous Nuclear RNA Transcripts in Mammalian Cells: Accentuation by 5,6-dichloro-1-$\beta $-D-ribofuranosylbenzimidazole [pp. 5750-5754]Oncogenic Transformation of Mammalian Cells in vitro with Split Doses of X-Rays [pp. 5755-5758]Dynein Binds to and Crossbridges Cytoplasmic Microtubules [pp. 5759-5763]Evolution of Eusociality in Termites [pp. 5764-5768]Effects of Opiates and Demographic Factors on DNA Repair Synthesis in Human Leukocytes [pp. 5769-5773]Increased Expression of a Eukaryotic Gene in Escherichia coli through Stabilization of Its Messenger RNA [pp. 5774-5778]Regional Assignment of the Steroid Sulfatase-X-Linked Ichthyosis Locus: Implications for a Noninactivated Region on the Short Arm of Human X Chromosome [pp. 5779-5783]Antibody-Induced Modulation of Friend Virus Cell Surface Antigens Decreases Virus Production by Persistent Erythroleukemia Cells: Influence of the Rfv-3 Gene [pp. 5784-5788]Isolation and Characterization of Transducing Phage Coding for $\sigma $ Subunit of Escherichia coli RNA Polymerase [pp. 5789-5793]Methylation of Chloroplast DNAs in the Life Cycle of Chlamydomonas [pp. 5794-5798]Gene Expression of an Escherichia coli Ribosomal RNA Promoter Fused to Structural Genes of the Galactose Operon [pp. 5799-5803]Assignment of Human $\beta $-, $\gamma $-, and $\delta $-globin Genes to the Short Arm of Chromosome 11 by Chromosome Sorting and DNA Restriction Enzyme Analysis [pp. 5804-5808]Fv-2 Locus Controls Expression of Friend Spleen Focus-Forming Virus-Specific Sequences in Normal and Infected Mice [pp. 5809-5812]Genetic Variability Maintained in a Finite Population under Mutation and Autocorrelated Random Fluctuation of Selection Intensity [pp. 5813-5817]Specific-Locus Test Shows Ethylnitrosourea to be the Most Potent Mutagen in the Mouse [pp. 5818-5819]DNA-Mediated Gene Transfer of a Circular Plasmid into Murine Cells [pp. 5820-5824]Antibodies to Epstein-Barr Virus-Determined Antigens in Normal Subjects and in Patients with Seropositive Rheumatoid Arthritis [pp. 5825-5828]Human Lymphocyte Antigens: Production of a Monoclonal Antibody that Defines Functional Thymus-Derived Lymphocyte Subsets [pp. 5829-5833]Dissociation and Exchange of the $\beta _{2}$-microglobulin Subunit of HLA-A and HLA-B Antigens [pp. 5834-5838]Amino Acid Sequence of an Immunoglobulin-Like HLA Antigen Heavy Chain Domain [pp. 5839-5842]Disassembly of Viral Membranes by Complement Independent of Channel Formation [pp. 5843-5847]Effect of Interchain Disulfide Bond on Hapten Binding Properties of Light Chain Dimer of Protein 315 [pp. 5848-5852]Structural Characterization of the Murine Fourth Component of Complement and Sex-Limited Protein and their Precursors: Evidence for Two Loci in the S Region of the H-2 Complex [pp. 5853-5857]Induction of Calcium Flux across the Rat Mast Cell Membrane by Bridging IgE Receptors [pp. 5858-5862]Crossreactive Mixed Lymphocyte Reaction Determinants Recognized by Cloned Alloreactive T Cells [pp. 5863-5866]Regulation of the Amplification C3 Convertase of Human Complement by an Inhibitory Protein Isolated from Human Erythrocyte Membrane [pp. 5867-5871]Evidence for a Two-Domain Structure of the Terminal Membrane C5b-9 Complex of Human Complement [pp. 5872-5876]Primary Defect of Insulin Receptors in Skin Fibroblasts Cultured from an Infant with Leprechaunism and Insulin Resistance [pp. 5877-5881]A Practicable Immunological Approach to Block Spermatogenesis without Loss of Androgens [pp. 5882-5885]Neoplastic Transformation of Epithelial Cells in Whole Mammary Gland in vitro [pp. 5886-5890]Retinoid Prevents Mammary Gland Transformation by Carcinogenic Hydrocarbon in Whole-Organ Culture [pp. 5891-5895]Enhancement of Hexose Uptake in Human Polymorphonuclear Leukocytes by Activated Complement Component C5a [pp. 5896-5900]Effect of Glucose/Sulfonylurea Interaction on Release of Insulin, Glucagon, and Somatostatin from Isolated Perfused Rat Pancreas [pp. 5901-5904]Biochemical and Morphologic Studies on Diabetic Rats: Effects of Sucrose-Enriched Diet in Rats with Pancreatic Islet Transplants [pp. 5905-5909]Spontaneous Tumors in Sprague-Dawley and Long-Evans Rats and in their F$_{1}$ Hybrids: Carcinogenic Effect of Total-Body x-Irradiation [pp. 5910-5913]Initiation of Plasma Prorenin Activation by Hageman Factor-Dependent Conversion of Plasma Prekallikrein to Kallikrein [pp. 5914-5918]Triene Prostaglandins: Prostaglandin D$_{3}$ and Icosapentaenoic Acid as Potential Antithrombotic Substances [pp. 5919-5923]Role of Thymidylate Synthetase Activity in Development of Methotrexate Cytotoxicity [pp. 5924-5928]Pyrolysis Products from Amino Acids and Protein: Highest Mutagenicity Requires Cytochrome P$_{1}$-450 [pp. 5929-5933]Changes in Plasma Lipoprotein Distribution and Formation of Two Unusual Particles after Heparin-Induced Lipolysis in Hypertriglyceridemic Subjects [pp. 5934-5938]Galactosamine-Induced Sensitization to the Lethal Effects of Endotoxin [pp. 5939-5943]Antigen-Induced Strain-Specific Autoantiidiotypic Antibodies Modulate the Immune Response to Dextran B 512 [pp. 5944-5947]Reactivation of Herpes Simplex Virus Type 2 from a Quiescent State by Human Cytomegalovirus [pp. 5948-5951]Social Gliding is Correlated with the Presence of Pili in Myxococcus xanthus [pp. 5952-5956]Apolipoprotein is Responsible for Neutralization of Xenotropic Type C Virus by Mouse Serum [pp. 5957-5961]Persistence of Circadian Rhythmicity in a Mammalian Hypothalamic ``Island'' Containing the Suprachiasmatic Nucleus [pp. 5962-5966]Specific Association of Neurotransmitter with Somatic Lysosomes in an Identified Serotonergic Neuron of Aplysia californica [pp. 5967-5971]Distribution of Active and Inactive Forms of Endorphins in Rat Pituitary and Brain [pp. 5972-5976]Widespread Distribution of Protein I in the Central and Peripheral Nervous Systems [pp. 5977-5981]Immunocytochemical Localization, in Synapses, of Protein I, an Endogenous Substrate for Protein Kinases in Mammalian Brain [pp. 5982-5986]Chronic Treatment with Lithium or Desipramine Alters Discharge Frequency and Norepinephrine Responsiveness of Cerebellar Purkinje Cells [pp. 5987-5991]Migration of Schwann Cells and Wrapping of Neurites in vitro: A Function of Protease Activity (Plasmin) in the Growth Medium [pp. 5992-5996]Blockage of Narcotic-Induced Dopamine Receptor Supersensitivity by Cyclo(Leu-Gly) [pp. 5997-5998]Localization of Neurophysin within Organelles Associated with Protein Synthesis and Packaging in the Hypothalamo-neurohypophysial System: An Immunocytochemical Study [pp. 5999-6003]Higher Molecular Weight Forms of Immunoreactive Somatostatin in Mouse Hypothalamic Extracts: Evidence of Processing in vitro [pp. 6004-6008]Intracellular Dye-Marked Enkephalin Neurons in the Magnocellular Preoptic Nucleus of the Goldfish Hypothalamus [pp. 6009-6011]Interrelationships between Ganglionic Acetylcholinesterase and Nonspecific Cholinesterase of the Cat and Rat [pp. 6012-6016]Membrane Potential Changes Caused by Thyrotropin-Releasing Hormone in the Clonal GH$_{3}$ Cell and their Relationship to Secretion of Pituitary Hormone [pp. 6017-6020]Correction: Amino Acid Sequence of Tyrosinase from Neurospora crassa [p. 6021]Correction: Methylation of Herpesvirus saimiri DNA in Lymphoid Tumor Cell Lines [p. 6021]Back Matter