Stability of rabbit globin and its messenger RNA in the mouse ovum

Preliminary 3. Miller, 0 L & Beatty, B R, Science 164(1969) 955. 4. Miller, 0 L & Bakken, A H, Acta endocrinol, suppl. 168 (1972) 155. 5. Olins. A L & Olins. D E. Science 183 (1974) 330. 6. Oudet, P, Gross-Bellard; M & Chambdn, P; Cell 4 (1975) 281. 7. Komberg, R D, Ann rev biochem 46 (1977) 931. 8. Mathis, D, Oudet, P & Chambon, P, Prog nucleic acid res mol biol 24 ( 1980) 1. 9. Franke, W W, Scheer, U, Trendelenburg, M F & Zentgraf, H, Cold Spring Harbor symp quant biol 42 (1978) 755. 10. Franke, W W, Scheer, U, Spring, H, Trendelenburg, M F & Zentgraf, H, The cell nucleus (ed H Busch) vol. 7, p. 49. Academic Press, New York (1979). 11. Griffith, J D, Science 187 (1975) 1202. 12. Derenzini. M. Hernandez-Verdun. D & Bouteille. M, Biol cell 4i (1981) 161. 13. - Submitted for publication. 14. Higgins, G H & Anderson, R M, Arch path01 12 (1931) 186. 15. Schmid, W & Sekeris, C E, FEBS lett 26 (1972) 109. 16. Derenzini, M, Pession-Brizzi, A, Bonetti, E & Novello. F. J ultrastruct res 67 (1979) 161. 17. Derenzini, M, Pession-Brizzi, A, Betts-Eusebi, C & Novello, F, J ultrastruct res 75 (1981) 229. 18. Derenzini, M, J ultrastruct res 69 (1979) 239. 19. Hemandez-Verdun, D, Derenzini, M & Bouteille, M, Biol cell 41 (1981) 17a. 20. - Chromosoma. In press. 21. Goessens, G & Lepoint, A, Biol cell 35 (1979) 211. 22. Scheer, U, Cell 13 (1978) 535. 23. - Eur j cell biol 23 (1980) 189. 24. Petrov, P, Raitcheva, E & Tsanev, R, Eur j cell biol 22 (1980) 708. 25. Lamb, M M & Daneholt, B, Cell 17 (1979) 835. 26. Finch, J T, Noll, M & Kornberg, R D, Proc natl acad sci US 72 (1975) 3320. 27. Scheer, U & Zentgraf, H, Chromosoma 69 (1978) 243. 28. Derenzini, M, Hernandez-Verdun, D & Bouteille, M, Biol cell 40 (1981) 147.

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Summary. Fertilized one-cell mouse ova were injected with rabbit globin messenger RNA, (mRNA) containing approximately equal quantities of (Y- and P-message. The half-lives of (Y-and p-globin were 8.2 and 7.0 h respectively. The half-lives of (Y- and /3globin messages were 8.8 and 9.5 h respectively. Approx. four times as much a- as fi-globin was present in the ova following the injections.

Im 1971, Gurdon et al. demonstrated that rabbit globin messenger RNA (mRNA) was accurately translated when microinjetted into the cytoplasm of Xenopus oocytes [ 11. These pioneering studies paved the way for the use of the Xenopus oocyte to study translation of a variety of mRNAs and also led to the use of the Xenopus oocyte to study transcription of specific DNA molecules [2]. In recent studies we have demonstrated that injected mRNA is translated by the mouse ovum [3] and that DNA is faithfully transcribed [4]. However, our initial studies suggested that the injected messages were not handled identically by the Xenopus oocyte and mouse ovum. We report here that indeed there exist significant differences between the Xenopus and the mouse in the stability of rabbit globin message following injection into the cytoplasm. Materials and Methods

Fertilized one-cell ova were obtained from superovulated C57x SJL hvbrid females. Following collection in Brinster’s medium [5], the ova were microinjetted as previously described [3]. Approx. 13 pl of rabbit globin mRNA (half a-globin and half /3-globin, J. Lingrel personal communication) at a concentration of 1 pg/pl were injected into the cytoplasm of each ovum and the ova were pooled. Proteins were labelled by incubating the injected ova in culture Copyright @ 1982 by Academic Press, Inc. medium containing a mixture of five 3H-labelled amino All rights of reproduction in any form reserved acids (leucine, lysine, phenylalanine, proline, tyrosine; 0014-4827/82/10@469-04$02.00/O sp. act. 75-120 Cilmmol; Amersham Searle). Incorporation of label into globin protein was determined followine senaration of the ovum nroteins bv twoStability of rabbit globin and its messenger dimensi&al klectrophoresis and removing the areas RNA in the mouse ovum of the gel corresponding to the location of globin [6]. A typical fluorograph is shown in fig. 1. The (Y- and H. Y. CHEN and R. L. BRINSTER, Laboratory of B-globin proteins were identified by their co-migration Reproductive Physiology, School of Veterinary Medi‘with no&radioactive globin markers [3]. The areas of cine, University of Pennsylvania. Philadelphia, PA the gel corresponding to globin protein were removed, placed in NCS (Amersham Searle) and water (9: 1). 19104. USA Received April 6, 1982 Accepted May 21, 1982

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Preliminary

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IEF

1. Fhrorograph of two-dimensional electrophoretic separation of proteins from 20 one-cell fertilized mouse ova injected with rabbit globin mRNA and labelled with [3H]amino acids for 15 h. Isoelectric focusing was from left (pH 7.5) to right (pH 4.5), and Fig.

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.

4.5

SDS polyacrylamide gel electrophoresis was from top to bottom. The position of a- and /3-globin is indicated by arrows. There is no radioactivity in this area when ova are not injected with the message.

Preliminary incubated for 16 h at 50°C and radioactivity mined by liquid scintillation counting.

deter-

Results

In the first series of experiments the stability of rabbit globin protein was determined by incubating ova injected with globin mRNA for 16 h in radioactive amino acids. The ova were then washed and placed in medium containing non-radioactive amino acids at 200 times the concentration of the labelled precursors. The ova were divided into three groups and assayed for labelled globin protein at 0, 4 and 8 h. On the basis of the disappearance of labelled globin protein from the ova during the 8 h chase period, the half-life of the protein was calculated. The results of six separate experiments are shown in table 1. There is no significant difference in the stability of Qglobin (Ta=8.2 h) and P-globin (T)=7.0 h). On the basis of the amounts of (Y- and pglobin present at 0 h, the relative amounts of each polypeptide synthesized were also determined and these values are shown in table 1. Approx. 4 times as much cy- as pglobin was present. In the second series of experiments the stability of the rabbit globin mRNA was determined. Ova were injected and randomTable

1. Half-lives of CY-and /I-rabbit globin synthesized from message injected into the fertilized mouse ovum Half-life (hours) Expt

a-Cilobin

@Globin

a-globin/ P-globin synthesized

1 2 3 4 2

10.4 8.2 5.8 10.3 8.4 6.3

5.6 5.9 4.8 11.1 7.0 7.7

1.1 2.6 6.7 4.4 4.2

Mean + SEM

8.2+0.8”

7.OkO.9”

a Means are not significantly different.

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Table 2. Half-lives of (Y- and /Gabbit globin mRNA injected into the fertilized mouse ovum

Expt

a-Globin

@Globin

a-Globin/ P-globin synthesized

1 2 3 4 5 Mean k SEM

6.4 8.9 10.9 7.2 10.5 8.8kO.9”

9.4 9.5

6.7 2.2

9.5

;.a

Half-life (hours)

ksto.

1”

4.9f 1.4

a Means are not significantly different.

ly divided into three groups. Labelling was begun in one of the groups at 4, 8, or 16 h following the completion of injections, and each group was labelled for a 12-h period. The amount of globin protein formed during the 12 h labelling period for each group was used as a measure of the mRNA remaining at each time following injection. From these values, the half-life of the message was calculated. The results of five separate experiments are shown in table 2. In two experiments the amount of /3-globin present was too low for accurate determination. This was a result of the smaller quantity of /3globin synthesized compared to a-globin. A comparison of a-globin and /3-globin present in these experiments again indicated that four times as much LYas p was present. The half-life of (Y- and @globin mRNA was approx. 9 h for both messages. Discussion

In Xenopus oocytes, the half-life of globin mRNA is long. Following injection of the message, the level of synthesis is only slightly reduced after 2 weeks and much of this reduction could be accounted for by the overall decrease in oocyte protein synthesis [7]. It has also been demonstrated that folExp Cell

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lowing globin message injection into the fertilized Xenopus egg, the message is still present and continues to be translated in differentiated tissue of the tadpole. Clearly the response of the fertilized mouse ovum to injected globin message is different. The stability is much less in these cells, where the message has an average half-life of approx. 9 h for both the CP and P-message. This more nearly approximates the stability of endogenous globin message in reticulocytes from which the message is isolated, where the effective half-life is only about 24 h [8]. In studies employing HeLa cells the half-life of duck globin message was also determined to be approx. 24 h [9]. The possibility exists that factors are present in the Xenopus oocyte and Xenopus fertilized egg which stabilize the globin message. These may be the same factors which serve to stabilize endogenous messages, since it is believed that many oocyte messages are long lived or are unavailable for translation (masked message). Whereas this may be a possibility in the oocyte, it seems less likely to apply to the Xenopus fertilized egg and early embryo, where message translation is increasingly active. Rather than being related to oocyte or embryo factors alone, the increased stability in Xenopus compared with mouse ova may be related, in part, to an inherent difference in amphibian and mammalian cytoplasmic factors. The similarity among the half-lives in the reticulocyte, HeLa cell and mouse ovum would lend weight to such a possible explanation. A second major difference in the response of the mouse ovum compared to Xenopus oocytes and ova following globin mRNA injection is the relative amount of (Y- and /Gpolypeptides synthesized. In Xenopus about five times as much /3- as cw-globin is synthesized. In our studies the overall ratio of (Y- to @globin present was E.rp Cell

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4.2kO.7 (nine values). This probably represents a difference in synthetic rates in the mouse egg since the half-lives of CY-and p- message are not significantly different, nor are the half-lives of the polypeptides significantly different. The possibility exists that the techniques for isolation (cell lysis and two-dimensional gel electrophoresis) could result in a dominance of one polypeptide over the other. However, it has generally been found that (Y rather than /3 is more subject to degradation and aggregation during isolation. What accounts for this apparent difference in synthetic rate is not clear, but it could be related to differences between the two messages in their secondary structure and availability of initiation codons [ 10, 111. The mouse ovum which has a rather low metabolic rate and protein synthesizing ability may be particularly sensitive to differences in message structure [12, 131. The Xenopus oocyte has proved to be a useful system for analyzing exogenous messages and DNA sequences. Whereas its large size makes it more convenient to manipulate than the mouse ovum, the response of the two systems to injected substances is different. In the case of globin, the mouse ovum clearly degrades the message much more rapidly, in a time more similar to the cell of origin. In addition the Xenopus and mouse ovum formed different ratios of (Y-and P-polypeptides from a message that contains approximately equal amounts of the respective messages. These findings suggest that the mouse system should provide a useful complement to the Xenopus system for analysing injected molecules. Furthermore the microinjection of mouse eggs should prove valuable in studying protein and nucleic acid metabolism of the egg and preovulatory stages of the oocyte.

Preliminary We thank Myma Trumbauer assistance. The rabbit globin supplied by Dr Jerry Lingrel. helpful advice and comments. from NIH grant HD 15477 8107172.

for excellent technical mRNA was generously We also thank him for Financial support was and NSF grant PCM-

References 1. Gurdon, J B, Lane, C D, Woodland, H R & Marbaix, G, Nature 233 (1971) 177. 2. Brown, D D & Gurdon, J B, Proc natl acad sci US 74 11977) 2064. 3. Bri&ter: R L, Chen, H Y, Trumbauer, M E & Avarbock. M R. Nature 283 (1980) 499. 4. Brinster, R L, Chen, H Y & Trumbauer, M E, Science 211 (1981) 396. 5. Brinster, R L, Growth, nutrition and metabolism of cells in culture. Academic Press, New York (1972). 6. Brinster, R L, Brunner, S, Joseph, X & Levey, I L, J biol them 254 (1979) 1927. 7. Gurdon, J B, Lingrel, J B & Marbaix, G, J mol biol 80 (1973) 539. 8. Gurdon, J B, Protein synthesis in reproductive tissue. Karolinska Institutet, Stockholm, Sweden (1973). 9. Stacey, D W & Allfrey, V G, Cell 9 (1976) 725. 10. Lockard, R E & RajBhandary, U L, Cell 9 (1976) IA7 , .,.

11. Pavlakis, G N, Lockard, R E, Vamvakopoulos, N, Rieser, L, RajBhandary, U L & Voumakis, J N, Cell 19 (1980) 91. 12. Mills, R M Jr & Brinster, R L, Exp cell res 47 (1967) 337. 13. Epstein, C J & Smith, S A, Dev bio133 (1973) 171. Received April 23, 1982 Accepted May 12, 1982

Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/82/1CKM73-07$02.00/O

Expression of human histocompatibility antigens on the surface of murine cells transformed by cosmid clones containing HLA genes P. LE BOUTEILLER,’ C. FOA,2 M. MALISSEN,’ P. GOLSTEIN,’ J. R. GALINDO: 2. MISHAL,’ D. CAILLOL’ and F. A. LEMONNIER,’ ‘Centre d’lmmunologie INSERM-CNRS de Marseille-Luminy, 13288 Marseille CPdex 9, and =INSERM U. 119, 13009 Marseille, France Summary. fibroblasts containing (TK) and Printed

Thymidine kinase-negative C3H mouse L (LMTK-) transformed with cosmid clones both herpes virus-derived thymidine kinase HLA class I genes were first selected in

in Sweden

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HAT (hypoxanthine, aminopterin, thymidine) medium and subsequently analyzed for the expression of human transplantation antigens. TK+-transformed cells expressing HLA class I molecules were characterized by surface radioimmunoassay, cytofluorimetric analysis and immunoperoxidase PAP technique at the light and electron microscopic levels, using a set of monoclonal antibodies. Comparisons were made with human B (Raji) and T (1301) lymphoblastoid cell lines which respectively express high and low levels of HLA molecules on their surface. The expression of HLA class I in association with murine gZmicroglobulin on the surface of transformed cells did not reduce the level of expression of H-2 molecules.

The HLA gene products of the human major histocompatibility complex (MHC) are of particular interest because of their role in transplantation and in many other immunological reactions [l]. HLA class I molecules are the usual targets for allogeneic (allograft rejection) or modified syngeneic (lysis of virally infected cells) cytolytic T lymphocytes. These and other properties justify the vast amount of work aiming at both identification of the genes coding for the different HLA class I molecules and definition of the biochemical structure of these molecules. HLA class I molecules consist of two polypeptides in non-covalent association: a highly polymorphic heavy chain (43000 D), encoded in the MHC, and a non-polymorphic light chain, p2-microglobulin (12000 D), not encoded in the MHC and highly preserved in different species [2]. However, due to the absence of informative intra HLA-recombinant material the analysis of HLA class I molecules has been difficult. The recent isolation [3] and sequence analysis [4] of a HLA class I gene and the possibility to introduce and express in murine fibroblast cells heterologous MHC genes [5] has opened new perspectives for the analysis of the HLA complex. An HLA class I probe derived from the sequenced gene [4] was used to isolate out of a human cosmid gene library those containing HLA class I genes. These Exp

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