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Kll4-4827/i9/oWo73+%02.C0/0
Experimental
LENS-SPECIFIC
mRNA
NEURAL IAIN
Institute
RETINA THOMSON,’
of Animal
Genetics,
Cell Research
122 (1979) 73-81
IN CULTURES AND
OF EMBRYONIC
PIGMENTED
EPITHELIUM
DAVID I. de POMERAI,’ JAMES and RUTH M. CLAYTON University
of Edinburgh,
CHICK
Edinburgh
F. JACKSON3
EH9 SJN,
Scotland
SUMMARY Messenger RNA (mRNA) from cultures of &day embryonic chick neural retina and pigmented epithelium has been characterised by cell-free translation and by hybridisation to complementary DNA (cDNA) which is complementary to the most abundant mRNAs isolated from the day-old chick lens. In neural retina cultures cell-free translation of total RNA gives detectable crystalhn products by 19 days in culture and the proportion of crystallin products gradually increases as the cultures progress, until at 42 days crystallins are the most prominent products. Hybridisation with cDNA showed that by 42 days in culture there is a 500-fold increase in the level of lens-specific mRNA relative to total mRNA. Most of this increase occurs during the final 12 days of culture. The levels of lens-specific mRNA also increase significantly in pigmented epithelium cultures-by 60 days in culture there is an IO-fold increase over the level in the starting material. The results provide evidence for transcriptional control of crystallin synthesis during culture.
During normal development of the chick, the lens of the eye is formed from a vesicle which is produced by invagination of the competent head ectoderm after induction by the eye cup. The lens consists entirely of two cell types: the lens epithelial cell, and the lens fibre cell which differentiates from it after a final mitosis. Lens cells are characterised by their ultrastructure and specific protein (crystallin) content and are unusual in that they can be formed via various alternative pathways from other eye tissues both in vivo and in vitro [l]. Two of these alternative pathways found in the chick are the ‘transdifferentiation’ systems whereby lens fibre-like cells can be detected after longterm cell culture of embryonic pigmented epithelium (PE) [2] or embryonic neural retina (NR) [3]. In both cases cells which resemble lens fibres in ultrastructure are localised in small groups (lentoid bodies) and
these have been shown to contain lenscharacteristic crystallins [2, 31. More recently we have quantitated the appearance of individual crystallins in transdifferentiating NR using biochemical and immunological techniques [4]. In these experiments very low levels of crystallins were found in freshly excised embryonic neural retina. After a few days in culture crystallins could no longer be detected, but by 2 weeks crystallins were again detectable and large amounts accumulated by the end of the culture period (6 weeks). 1 Present address: Department of Biological Sciences, Napier College of Commerce and Technology, Colinton Road, Edinburgh EHlO 5DT, Scotland. * Present address: Department of Zoology, University of Nottingham, University Park, Nottingham NG7 2RD, UK. 3 Present address: Columbia University, College of Physicians and Surgeons, Institute for Cancer Research, 701 West 168th Street, New York, NY 10032, USA.
74
Thomson et (11.
In order to gain insight into the molecular mechanisms involved in transdifferentiation (or indeed normal differentiation), it is important to discover how the synthesis and accumulation of large amounts of crystallins is controlled. Several studies have shown the presence of small amounts of crystallins in non-lens ocular tissues of the chick, e.g. iris and cornea [5, 61, and embryonic NR [4]. These tissues must either contain crystallin messenger RNA (mRNA) or have acquired small amounts of crystallin from the lens. Recently we have shown by molecular hybridisation that both neural retina and pigmented epithelium from g-day embryonic chicks contain low levels of putative crystallin mRNA sequences [7]. We have also detected large amounts of crystallins produced by heterologous cell-free translation of mRNA isolated from 42-day cultures of embryonic NR, whereas no crystallin products were detectable when mRNA from freshly excised embryonic NR was translated [g]. Several mechanisms could be put forward to explain these results, e.g.: (1) There could be preferential translation of pre-existing mRNA to produce large amounts of crystallins (during culture); (2) increased transcription of crystallin genes may occur to produce large amounts of crystallin mRNAs; (3) the stability of mRNAs may change during the culture period. In this paper we have used the techniques of cell-free translation and molecular hybridisation with complementary DNA (cDNA) to analyse mRNA isolated from embryonic NR cultures at various stages of culture to try to distinguish between these and other possible mechanisms of crystallin gene expression. This type of approach has been used in a variety of developmental systems where there is a large increase in the synthesis of a particular protein. These include ovalbumin [9], haemo-
globin [IO] and silk moth fibroin [ 111. and in general the results have favoured transcriptional control of gene expression. Embryonic PE cultures are less suitable for a similar study as transdifferentiation takes longer and there are fewer lentoids produced. Also there is a lesser accumulation of crystallins in PE cultures as opposed to NR cultures (Pritchard & de Pomerai, unpublished results). We have, however, compared the amounts of lens-specific mRNA in the RNA isolated from the terminal stage of PE cultures (after 60 days in culture) with the amounts in freshly excised embryonic PE, in order to compare the levels of lensspecific gene expression in two different culture systems. MATERIALS
AND
METHODS
Day-old chicks and fertile eggs were obtained from Ross Poultry Ltd, Newbridge, Midlothian, Scotland. [3H]dCTP and [YS]methionine were purchased from The Radiochemical Centre, Amersham, Bucks. Micrococcal nuclease was obtained from Worthington Enzymes, London and St nuclease from Sigma, London.
Cell culture techniques Neural retinae from 8-dav chick embrvos were diesected out and prepared for cell culture as described by Okada et al. [3]. The dissociated cells were then cultured as previously described [4]. Pigmented epithelium was dissected from 8-day chick embryos, and prepared for cell culture as described by Eguchi & Okada [2]. The dissociated cells were then cultured under conditions identical to those used for the NR cultures. “.-
Preparation
of RNA
Total polysomal RNA was isolated from day-old chick lenses aS ,previously described [8]. Poly(A)-containing RNA was then separated by two cycles of oligo-dTcellulose chromatography. Using this procedure polysomal RNA consisted approx. 80% of total cytoplasmic RNA. For preparation of RNA from culture material, cultures at various stages were washed in saline, homogenised in 0.35 M sucrose, 10 mM Tris pH 7.5, 50 mM KCI, 1.5 mM MgClz and centrifuged at 10000 g for 10 min at 4°C. Total cytoplasmic RNA was prepared from the supematants by extraction several times with ohenol/chloroform (l/l bv vol), 0.05 % 8hydroxyquinoline ‘and collected by ethanol precipitation.
Lens mRNA Cell-free trunslation of RNA Messenger-dependent lysate was prepared from rabbit reticulocytes according to the method of Pelham & Jackson [ 121 with the exception that incubation of the lysate with micrococcal nuclease was for 5 min at 30°C. Translation assays were carried out exactly as described by these authors. One pg of mRNA or 10 pg of total RNA was added to a 50 ~1 translation mix. Using day-old chick lens RNA an approximately linear increase in incorporated radioactivity was obtained by adding up to 5 pg of mRNA or 20 pg of total RNA to the translation mix.
Polyacrylamide gel electrophoresis Proteins synthesised in the cell-free system were analysed by SDS-polyacrylamide gel electrophoresis on discontinuous slab gels. The gel was based on the mixture of Laemmli [13] and had a linear IO-20% acrylamide gradient, which was stabilised during pouring andpolymerisation by the addition of 20% glycerol. Marker crystallin standards and up to 10 ~1 of the translation mix was applied to individual slots and the gel run overnight at a constant voltage of 100 V. The gel was then either first stained as previously described [14] or subjected directly to fluorography to detect labelled proteins as described by Bonner & Laskev rlS1 usina me-flashed X-rav film to give a quantitative-response io radioactive disintegrations [ 161.
Preparation of cDNA To prepare cDNA complementary to the most abundant sequences present in the day-old chick lens, cDNA was first prepared from polyA-containing RNA isolated from day-old chick polysomes as previously described 171. Preparation of the fraction of cDNA which is complementary to the most abundant lens sequences was carried out by modification of the method of Hastie & Bishop [17] as follows. Seventy nanogram of lens cDNA was reacted with 3.5 ua of lens mRNA in 20 ~1 of formamide hybridisation &ffer (0.5 M NaCl. 25 mM HEPES. DH 6.8.0.5 mM EDTA. 50 mM EDTA, 50% (v/v) deibnised formamide) in a siliconised capillary tube to a Rot of 1.5~ 10-r (10 times the R,,tf of the most abundant class of sequences r71). The contents of the canillarv were flushed out into 230 ~1 of nuclease assay buffer (50 mM sodium acetate, pH 4.5, 2.8 mM ZnSO,, 0.1 M NaCl, 20 pg/ml denatured rat DNA) and 20 units of Sl nuclease added. The mixture was incubated for 1 h at 37°C and the reaction stopped by extraction with an equal volume of phenol : chloroform (1 : 1) 0.05 % 8hvdroxvquinoline. The phenol layer was re-extracted with iM ~1 H,O and the supematants combined. RNA was destroyed by treatment with 0.3 N NaOH for 1 h at 3PC. The solution was neutralised and cDNA collected by passage through a Sephadex G-50 column. The cDNA isolated was reacted again with lens mRNA and the procedure repeated. After two cycles of hybridisation the yield of abundant cDNA was about 30 % of the input cDNA. Alkaline sucrose gradients showed the abundant cDNA to be of similar length to a sample of
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75
the original cDNA (300-350 nucleotides). Abundant cDNA prepared in this way is lens-specific, as judged bv its hvbridisation to lens mRNA with aR,,ft of 2.58x I&’ mol-set/l and failure to hybridise to embryo body or smooth muscle mRNA at R,,t values as high as 1X 1o;Lmol-sec/l[7].
Hybridisation
reactions
Appropriate volumes of RNA and abundant cDNA solutions in sterile distilled water were mixed, lyophilised and dissolved in formamide hvbridisation buffer as above. The ratio of mRNA to-cDNA in these mixtures was 20: 1 or greater. Aliauots of the hvbridisation mixture (I-10~@I) were sealed in siliconised glass capillaries. The capillaries were heated at 65°C for 5 mm, then incubated at 43°C for various times. The contents of the capillaries were flushed out with 0.25 ml of nuclease assay buffer (50 mM sodium acetate pH 4.5, 2.8 mM ZnSO,, 0.1 M NaCl, 20 pg/ml denatured rat DNA) and frozen. The proportion of cDNA in hybrid was determined by addition of 20 units of Sl nuclease and calculating the proportion of acidsoluble radioactivity after incubation for 1 h at 37°C. In control experiments this amount of Sl nuclease was shown to be capable of completelv degrading the amount of cDNA used in the-presence of 1 mg/ml E. co/i RNA. Some 2-3% of dav-old lens cDNA and IO-1 1% of abundant cDNA was resistant to Sl nuclease digestion in the absence of complementary sequences (double-stranded sequences are conserved during the preparation of abundant cDNA). Values for percentage hybridisation in the presence of complementary RNA sequences were corrected to allow for this.
RESULTS Hybridisation reactions using total RNA
For most of the culture stages studied only a small amount of material was available and so in general only total cytoplasmic RNA was prepared from individual stages. It was therefore necessary to see if the vast excess of rRNA present affected the behaviour of the mRNA in cell-free translation and hybridisation experiments. We have shown that total lens polysomal RNA and polysomal mRNA selected by oligo(dT)cellulose chromatography are translated in the cell-free system to give very similar crystallin profiles [8]. Fig. 1 shows the result of hybridising abundant cDNA to (a) day-old lens polysomal mRNA, and (b) Exp Cell
Res 122 (1979)
76
Thomson
Table 1. Dosage
et rrl. oflens-spc)c(fic
sequences
Source of material
RNA sample
Rdt
Day-old chick lens Day-old chick lens S-day embryo NR S-day embryo PE 30-day NR cultures 42-day NR cultures 60-day PE cultures
Total polysomal RNA Polysomal mRNA Cytoplasmic mRNA Cytoplasmic mRNA Total cytoplasmic RNA Total cytoplasmic RNA Total cytoplasmic RNA
2.77x 2.58x IX 6.05x 8.56X I .89x 7.20x
Dosage 10-Z lo-’ 102” IO’ 10-l 10-l 10-l
0.93 I 0.00026” 0.00043 0.030 0.136 0.035
R,,t& values were calculated using a least-squares programme which provided a single component curve which was the best fit for the data points. In reactions where total RNA was used, it was assumed that I % of the input RNA was participating in the reaction. The values for the dosage of lens-specific sequences are calculated relative to the level of crystallin mRNA sequences (i.e. those which are complementary to abundant cDNA) present in day-old chick lens polysomal mRNA, which has been given the value 1. 0 These values are estimated, as the hybridisations with S-day embryo NR mRNA did not reach saturation levels even at the highest R,,t values available.
day-old lens total RNA in conditions of mRNA excess. As 1% of the total RNA was selected as mRNA by oligo(dT)cellulose chromatography the Rot values for the total RNA reaction are calculated on the basis that 1% of the input RNA is participating in the reaction. It is clear that the
presence of rRNA has little or no effect on the hybridisation reaction. R,,tl values were calculated from both sets of data points using a least squares programme which provided the best fit for a single component curve [17] and these are shown in table 1. The values are very similar and cer-
7
1. Abscissa: log Rot; ordinate: % hybridisation. O-0, mRNA; m-M, total RNA. Day-010 ChicK lens polysomal mRNA (selected by two cycles of oligo(dT)-cellulose chromatography) or total polysomal RNA hybridised to abundant cDNA to various R,r values.
trll
Cell
KC\
122 (IY7Y)
23
30
35
42
2. Abscissa: time in cell culture (days); ordinate: identification and mol. wt of products which comigrate with crystallin subunits. Ten pg of total cytoplasmic RNA from NR cultures at various stages of development were translated in the cell-free system and the products of translation detected as described. Approximately equal amounts of radioactivity were added to each slot of the gel. The fluorograph was developed after a time sufficient to detect only the most prominent products.
Fig.
Fig.
12 19
Lens mRNA
3. Abscissa: log R,r: ordinate: % hvbridised. (Z&) O-O, Day-old chick lens polysom; mRNA; V-V. total cvtonlasmic RNA from 42-dav cultures of 8-day embryonic-NR; X-X, total cytopiasmic RNA from 30-day cultures of 8-day embryonic NR; W.-W, l -a total cytoplasmic mRNA from 8-day embryonic NR (data from two experiments shown). (Bottom) B-H, Total cytoplasmic RNA from 23-day cultures of 8-day embryonic NR; O---O, total cytoplasmic RNA from 19-day cultures of 8-day embryonic NR. RNA samples from day-old chick lens, 8-day embryonic NR or from culture material were hybridised to abundant cDNA to various R,t values. The solid lines are not computer fits to the data. The curves for lens and embryonic NR are shown for comparison. Fig.
tainly within the errors inherent in the experiment. Since abundant cDNA hybridises to day-old chick lens mRNA with kinetics very similar to those shown by the fastest hybridising component in an experiment in which total cDNA from day-old chick lens was used [7], it may be used to probe for lens specific sequences in total RNA preparations.
in cultured cells
11
Crystallin mRNAs in NR cultures We have shown that translatable crystallin mRNA is present in large amounts in 42 day cultures of g-day chick embryo NR [8]. In order to follow the appearance of crystallinspecific mRNA during the course of these cultures, we have prepared total cytoplasmic RNA from cultures harvested at various stages of development. The products of cell-free translation of the various RNAs were analysed on an SDS-polyacrylamide gel and a fluorograph of the labelled products is shown in fig. 2. In the early stages of the culture only a few proteins are synthesised in detectable amounts and none of these co-migrate with crystallins. By 19 days in culture proteins comigrating with crystallins, particularly ycrystallin and a P-crystallin with mol. wt 34000 can be detected and they gradually become more prominent at subsequent culture stages, so that by 42 days they represent most of the detectable products. We have shown previously that traces of both crystallins [4] and lens-specific mRNA [7] are present in freshly excised g-day embryo NR. Since translatable crystallin mRNAs become more prominent as the cultures progress, it seems likely that lens-specific mRNA as measured by hybridisation to abundant cDNA, should also increase. This is shown in tig. 3. During the early stages of the culture no hybridisation to abundant cDNA could be detected at a Rot up to 1X IO’. At 19 and 23 days in culture some lens-specific sequences were detected but only at very low concentrations. Some cDNA remained single-stranded even at the highest Rot values achievable in these experiments. (The Rot curve of RNA from freshly excised NR was constructed using data from an experiment in which mRNA was used, and so greater R,t values were achieved.) It is clear that most of the inExp CdRes
122(1979)
12 0
-
/
6
P
1 alImB -
=)
d.
Fig. 5. Abscissu: slice no.; ordinclre: radioactivity (cpm). A sample of the cell-free translation products of cytoplasmic RNA from 60-day PE cultures was immunoprecipitated with anti-total crystallin antiserum and analysed on an SDS gel as described previously [8]. The gel was sliced into 1 mm slices and the radioactivity in each determined. The migration of marker crystallins in a parallel slot of the gel is also shown.
when compared with freshly excised embryonic NR, and by 42 days a further 5-fold increase is seen (table 1). By this terminal stage the levels of these lens-specific sequences have reached approx. 10% of the levels found in day-old chick lens polysomal mRNA.
Cvystallins and crystallin mRNAs in PE cultures Fig. 4. An SDS-polyacrylamide gel was run as de- PE cultures are slower to develop than NR scribed in Materials and Methods. (a), (h) Stained protein bands; (c), (d) fluorograph of dried gel. (a) cultures and fewer lentoids develop in the Soluble proteins from 42-day cultures of 8-day em- terminal stages and so it would be expected bryonic NR; (b) soluble proteins from 60-day cultures that less crystallin would accumulate during of I-day embryonic PE; (c) cell-free translation prodthe course of the culture. A comparison of ucts of total cytoplasmic RNA from 42-day cultures of 8-day embryonic NR; (d) cell-free translation prod- the soluble proteins from the terminal ucts of total cytoplasmic RNA from 60-day cultures of stages of embryonic NR and PE cultures on 8-day embryonic PE. The migration of the major chick crystallin subunits in a parallel gel is shown for com- an SDS-acrylamide gel (fig. 4) shows that parison. whereas large amounts of crystallin have accumulated in the NR culture, proteins cocrease in lens-specific mRNA (relative to migrating with identifiable crystallins are total mRNA) is found during the later less prominent in the soluble protein from stages of the culture. A comparison of the the PE culture. Furthermore large amounts Rot* values shows that by 30 days in cul- of crystallin products are seen when mRNA ture lens-specific mRNAs have increased from 42-day NR cultures is translated in the relative to total mRNA by approx. lOO-fold cell-free system. Translation of mRNA tlvp
(‘r/l
Ke.s 122 (1979)
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Lens mRNA
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logR,t; ordinate: % hybridised. RNA samples from day-old chick lens, &day embryonic PE or culture material were hybridised to abundant cDNA to various R,r values. The solid lines are not computer fits to the data. O-O, Day-old chick lens polysomal mRNA; V-V, total cytoplasmic RNA from 60-day cultures of I-day embryonic PE; W-W, total cytoplasmic mRNA from 8-day embryonic PE.
Fig. 6. Abscissa:
from 60-day PE cultures gave a number of products, few of which co-migrate with crystallins (fig. 4). Previously, we have confirmed that a large proportion of the products of cell-free translation of 42-day NR culture mRNA were crystallins, by immunoprecipitations with anticrystallin antibodies [8]. Fig. 5 shows the result if a similar experiment using cell-free translation products of RNA from 60-day PE cultures. Only a very small proportion of the TCAprecipitable radioactivity was precipitated using antitotal chick crystallin antiserum (6 %, as opposed to 4-5 % in control experiments using non-immune serum). When run on an SDS-acrylamide gel a proportion of the radioactivity in the immunoprecipitate co-migrated with crystallin subtlnits. Some radioactivity is also seen in the high molecular weight region of the gel. The identity of this material is unclear but the most likely possibility is that these are lens membrane proteins, since lens membrane components run in this portion of the gel and the antiserum used has some specificity towards 6-791810
in cultured
cells
79
membrane proteins (Odeigah & Clayton, unpublished). However, by hybridisation to abundant cDNA, the amount of lens-specific mRNA present in 60-day PE cultures showed an 80-fold increase over the levels of these sequences present in freshly excised pigmented epithelium (fig. 6, table 1). Relative to total mRNA in each case, this is close to the level reached by NR cultures at 30 days of development. This is well short of the levels reached by NR cultures by the terminal stage and approx. 1% of the level of these sequences in day-old chick lens polysomal
mRNA.
DISCUSSION Whilst the cDNA complementary to the abundant mRNA sequences in the day-old lens has in this paper been referred to merely as lens-specific, there is now very good evidence that it is complementary only to crystallin mRNAs. This evidence can be summarised as follows: (1) The kinetic complexity of the cDNA suggests that it corresponds to only a small number (approx. 4) of different mRNAs [7]. (2) When day-old chick lens soluble proteins are separated on an SDS-acrylamide gel 7-9 prominent bands are seen and these are all identifiable crystallins [ 141. (3) Similarly, when day-old chick lens mRNA is translated in a cell-free system only crystallins are prominent products [8]. (4) The amounts of mRNA sequences complementary to the abundant cDNA increase markedly in two different cell culture systems where the amounts of both crystallins and translatable crystallin mRNAs are known to be increasing (this paper). Absolute proof of the nature of the abundant cDNA would require isolation and unambiguous identification of individual mRNAs after hybridisation to abunExp Cell Res 122 (1979)
dant cDNA, but any conclusion other than that the abundant cDNA is in fact crystallinspecific cDNA seems highly unlikely. The possibilities that (1) abundant mRNA _sequences may not include those coding for minor crystallins, and (2) cross-hybridisation between related but distinct mRNAs, may explain the discrepancy between the number of mRNA sequences represented in abundant lens cDNA and the number of crystallin protein subunits. In fact, hybridisation of 6-crystallin mRNA to its cDNA transcript occurs with kinetics which show a single sequence [19] while the protein consists of at least two similar but distinct subunits [ 14, 181. Under the conditions used here a single purified mRNA (globin mRNA) reacts with its cDNA copy over a range of 2.5 units on the log R,,t scale [20] and the lens mRNA-abundant cDNA reaction occurs over a similar range. If, in the heterologous reactions with RNA from NR and PE cultures, the relative concentrations of individual crystallin mRNAs were very different from those in day-old chick lens mRNA, the reaction would no longer occur as a single kinetic component. The hybridisation data from NR and PE cultures cannot be distinguished from that expected for a single component, but concentration differences of less than about ten-fold would not be detected. Since the plateau levels reached in the heterologous hybridisation reactions are similar to those reached in the homologous reactions then most or all of the mRNA sequences which are present in the day-old lens and complementary to crystallin cDNA are also present in the mRNA from the cultured material. In this paper two independent techniques show that crystallin-specific mRNA is increasing during culture of embryonic NR
and PE. It is important to correlate these results with the amounts of crystallin protein accumulated during the culture period. Using haemagglutination inhibition assays, de Pomerai et al. [4] have quantitated crystallin accumulation at various stages during culture of embryonic NR. Relative to the levels of crystallin mRNA or crystallin protein present in the day-old chick lens, the accumulation of crystallin specific mRNA and protein show similar patterns. A large increase occurs in both cases over the course of the culture period, mainly during the later stages. The most obvious interpretation of this data is that transdifferentiation in NR cultures is accompanied by transcription of crystallin genes to produce increasing amounts of crystallin mRNAs which are in turn translated. Crystallins accumulate until they account for a substantial proportion of the newly synthesised [8] and this paper) and accumulated [4] protein. We have not examined the levels of crystallin-specific mRNA in nuclear RNA, or the rate of mRNA breakdown, to see whether some post-transcriptional event could account for the increases seen in cytoplasmic mRNA. As both the number of cells and the amounts of crystallinspecific mRNA increase substantially during the culture it is unlikely that such a mechanism could contribute significantly to the overall sequence of events. Transcriptional control mechanisms have been proposed for other culture systems where differentiation of a particular cell type is occurring, e.g. for globin mRNA in cultured Friend erythroleukaemia cells [21]. In cultures of chick embryo muscle cells Paterson & Bishop [22] found no change in the total mRNA complexity during myogenesis (approx. 17000 sequences), but found large increases in the amounts of a small number of mRNAs. At the same time
Lens mRNA
there was increased synthesis of a small number of proteins when mRNA was isolated from the cultures and translated in a cell-free system. The total complexity of mRNA from lens cells is thought to be somewhat less [7] than that found in these muscle cells and in other chick tissues [23]. Perhaps this is because the lens consists of two cell types and is metabolically somewhat inactive. While a transcriptional control mechanism for crystallin gene expression in NR cultures is the most obvious interpretation of the data, other secondary levels of control are not excluded. There is good evidence that the accumulation of individual crystallins in NR and lens epithelium cultures is independently regulated [4]. A suitable assay for individual mRNAs would be required to determine whether this noncoordinate regulation is reflected at the mRNA level. The possibility that individual mRNAs are translated in vitro at differing efficiencies precludes the use of heterologous cell-free translation for such an analysis. However, DNA probes complementary to individual mRNA species provide a means to study these questions. Experiments to this end are currently in progress. We are grateful to Ross Poultry Ltd., Newbridge, Midlothian, for the supply of day-old chicks and fertile eggs. We thank Christine Wilkinson for skilled technical assistance and Drs R. Williamson and D. E. S. Truman for discussions. The work was supported by grants from the MRC and the Cancer Research Campaign. J. F. J. was supported by a postdoctoral research fellowship from Fight for Sight, Inc., New York City.
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REFERENCES 1. Clayton, R M, Stem cells and homeostasis (ed B I Lord, C J Potten & R J Cole), p. 115. Cambridge University Press, London (1978). 2. Euguchi, G & Okada, T S, Proc natl acad sci US 70(1973)1495. 3. 4.
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Okada, T S, Itoh, Y, Watanabe, K & Eguchi, G, Dev biol45 (1975) 321. de Pomerai, D I, Pritchard, D J & Clayton, R M, Dev biol60 (1977) 416. Clayton, R M, Campbell, J C & Truman, D E S, Exp eye res 7 (1%8) Il. Bours, J & van Doorenmaalen, W J, Exp eye res 13 (1972) 236. Jackson, J F, Clayton, R M, Williamson, R, Thomson, I, Truman, D E S & de Pomerai, D I, Dev biol65 (1978) 383. Thomson, I, Wilkinson, C E, Jackson, J F, de Pomerai, D I, Clayton, R M, Truman, D E S & Williamson, R, Dev bio165 (1978) 372. Harris, S E, Rosen, J M, Means, A R & O’Malley, B W, Biochemistry 14 (1975) 2072. Ross, J, Gielen, J, Packman, S I, Kawa, Y & Leder, P, J mol biol87 (1974) 697. Suzuki, Y & Suzuki, E, J mol biol88 (1974) 393. Pelham, H R B & Jackson, R J, Eur j biochem 67 (1976) 247. Laemmli, U K, Nature 227 (1970) 680. Thomson, I, Wilkinson, C E, Bums, A T H, Truman, D E S & Clayton, R M, Exp eye res 26 (1978) 351. Bonner, W M & Laskey, R A, Eur j biochem 46 (1974) 83. Laskey, R A & Mills, A 0, Eurj biochem 56 (1975) 335.
17. Hastie, N D & Bishop, J 0, Cell 9 (1976) 761. 18. Reszelbach, R, Shinohara, T S & Piatigorsky, J, Exp eye res 25 (1977) 583. 19. Zelenka, P & Piatigorsky, J, Exp eye res 22 (1976) 115. 20. Young, B D, Harrison, P R, Gilmour, S, Bimie, G D, Hell, Ai Humphries, S & Paul, J, J mol biol 84(1974)555.
71 -.. Amu, H, Voloch, Z, Bastos, R & Levy, S, Cell 8
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Received August 3 1, 1978 Revised version .__ . received .- .--- March 12, 1979 Accepted March 15, 1979
Exp Cd/
Re.\ 122 (1979)
Kll4-4827/i9/oWo73+%02.C0/0
Experimental
LENS-SPECIFIC
mRNA
NEURAL IAIN
Institute
RETINA THOMSON,’
of Animal
Genetics,
Cell Research
122 (1979) 73-81
IN CULTURES AND
OF EMBRYONIC
PIGMENTED
EPITHELIUM
DAVID I. de POMERAI,’ JAMES and RUTH M. CLAYTON University
of Edinburgh,
CHICK
Edinburgh
F. JACKSON3
EH9 SJN,
Scotland
SUMMARY Messenger RNA (mRNA) from cultures of &day embryonic chick neural retina and pigmented epithelium has been characterised by cell-free translation and by hybridisation to complementary DNA (cDNA) which is complementary to the most abundant mRNAs isolated from the day-old chick lens. In neural retina cultures cell-free translation of total RNA gives detectable crystalhn products by 19 days in culture and the proportion of crystallin products gradually increases as the cultures progress, until at 42 days crystallins are the most prominent products. Hybridisation with cDNA showed that by 42 days in culture there is a 500-fold increase in the level of lens-specific mRNA relative to total mRNA. Most of this increase occurs during the final 12 days of culture. The levels of lens-specific mRNA also increase significantly in pigmented epithelium cultures-by 60 days in culture there is an IO-fold increase over the level in the starting material. The results provide evidence for transcriptional control of crystallin synthesis during culture.
During normal development of the chick, the lens of the eye is formed from a vesicle which is produced by invagination of the competent head ectoderm after induction by the eye cup. The lens consists entirely of two cell types: the lens epithelial cell, and the lens fibre cell which differentiates from it after a final mitosis. Lens cells are characterised by their ultrastructure and specific protein (crystallin) content and are unusual in that they can be formed via various alternative pathways from other eye tissues both in vivo and in vitro [l]. Two of these alternative pathways found in the chick are the ‘transdifferentiation’ systems whereby lens fibre-like cells can be detected after longterm cell culture of embryonic pigmented epithelium (PE) [2] or embryonic neural retina (NR) [3]. In both cases cells which resemble lens fibres in ultrastructure are localised in small groups (lentoid bodies) and
these have been shown to contain lenscharacteristic crystallins [2, 31. More recently we have quantitated the appearance of individual crystallins in transdifferentiating NR using biochemical and immunological techniques [4]. In these experiments very low levels of crystallins were found in freshly excised embryonic neural retina. After a few days in culture crystallins could no longer be detected, but by 2 weeks crystallins were again detectable and large amounts accumulated by the end of the culture period (6 weeks). 1 Present address: Department of Biological Sciences, Napier College of Commerce and Technology, Colinton Road, Edinburgh EHlO 5DT, Scotland. * Present address: Department of Zoology, University of Nottingham, University Park, Nottingham NG7 2RD, UK. 3 Present address: Columbia University, College of Physicians and Surgeons, Institute for Cancer Research, 701 West 168th Street, New York, NY 10032, USA.
74
Thomson et (11.
In order to gain insight into the molecular mechanisms involved in transdifferentiation (or indeed normal differentiation), it is important to discover how the synthesis and accumulation of large amounts of crystallins is controlled. Several studies have shown the presence of small amounts of crystallins in non-lens ocular tissues of the chick, e.g. iris and cornea [5, 61, and embryonic NR [4]. These tissues must either contain crystallin messenger RNA (mRNA) or have acquired small amounts of crystallin from the lens. Recently we have shown by molecular hybridisation that both neural retina and pigmented epithelium from g-day embryonic chicks contain low levels of putative crystallin mRNA sequences [7]. We have also detected large amounts of crystallins produced by heterologous cell-free translation of mRNA isolated from 42-day cultures of embryonic NR, whereas no crystallin products were detectable when mRNA from freshly excised embryonic NR was translated [g]. Several mechanisms could be put forward to explain these results, e.g.: (1) There could be preferential translation of pre-existing mRNA to produce large amounts of crystallins (during culture); (2) increased transcription of crystallin genes may occur to produce large amounts of crystallin mRNAs; (3) the stability of mRNAs may change during the culture period. In this paper we have used the techniques of cell-free translation and molecular hybridisation with complementary DNA (cDNA) to analyse mRNA isolated from embryonic NR cultures at various stages of culture to try to distinguish between these and other possible mechanisms of crystallin gene expression. This type of approach has been used in a variety of developmental systems where there is a large increase in the synthesis of a particular protein. These include ovalbumin [9], haemo-
globin [IO] and silk moth fibroin [ 111. and in general the results have favoured transcriptional control of gene expression. Embryonic PE cultures are less suitable for a similar study as transdifferentiation takes longer and there are fewer lentoids produced. Also there is a lesser accumulation of crystallins in PE cultures as opposed to NR cultures (Pritchard & de Pomerai, unpublished results). We have, however, compared the amounts of lens-specific mRNA in the RNA isolated from the terminal stage of PE cultures (after 60 days in culture) with the amounts in freshly excised embryonic PE, in order to compare the levels of lensspecific gene expression in two different culture systems. MATERIALS
AND
METHODS
Day-old chicks and fertile eggs were obtained from Ross Poultry Ltd, Newbridge, Midlothian, Scotland. [3H]dCTP and [YS]methionine were purchased from The Radiochemical Centre, Amersham, Bucks. Micrococcal nuclease was obtained from Worthington Enzymes, London and St nuclease from Sigma, London.
Cell culture techniques Neural retinae from 8-dav chick embrvos were diesected out and prepared for cell culture as described by Okada et al. [3]. The dissociated cells were then cultured as previously described [4]. Pigmented epithelium was dissected from 8-day chick embryos, and prepared for cell culture as described by Eguchi & Okada [2]. The dissociated cells were then cultured under conditions identical to those used for the NR cultures. “.-
Preparation
of RNA
Total polysomal RNA was isolated from day-old chick lenses aS ,previously described [8]. Poly(A)-containing RNA was then separated by two cycles of oligo-dTcellulose chromatography. Using this procedure polysomal RNA consisted approx. 80% of total cytoplasmic RNA. For preparation of RNA from culture material, cultures at various stages were washed in saline, homogenised in 0.35 M sucrose, 10 mM Tris pH 7.5, 50 mM KCI, 1.5 mM MgClz and centrifuged at 10000 g for 10 min at 4°C. Total cytoplasmic RNA was prepared from the supematants by extraction several times with ohenol/chloroform (l/l bv vol), 0.05 % 8hydroxyquinoline ‘and collected by ethanol precipitation.
Lens mRNA Cell-free trunslation of RNA Messenger-dependent lysate was prepared from rabbit reticulocytes according to the method of Pelham & Jackson [ 121 with the exception that incubation of the lysate with micrococcal nuclease was for 5 min at 30°C. Translation assays were carried out exactly as described by these authors. One pg of mRNA or 10 pg of total RNA was added to a 50 ~1 translation mix. Using day-old chick lens RNA an approximately linear increase in incorporated radioactivity was obtained by adding up to 5 pg of mRNA or 20 pg of total RNA to the translation mix.
Polyacrylamide gel electrophoresis Proteins synthesised in the cell-free system were analysed by SDS-polyacrylamide gel electrophoresis on discontinuous slab gels. The gel was based on the mixture of Laemmli [13] and had a linear IO-20% acrylamide gradient, which was stabilised during pouring andpolymerisation by the addition of 20% glycerol. Marker crystallin standards and up to 10 ~1 of the translation mix was applied to individual slots and the gel run overnight at a constant voltage of 100 V. The gel was then either first stained as previously described [14] or subjected directly to fluorography to detect labelled proteins as described by Bonner & Laskev rlS1 usina me-flashed X-rav film to give a quantitative-response io radioactive disintegrations [ 161.
Preparation of cDNA To prepare cDNA complementary to the most abundant sequences present in the day-old chick lens, cDNA was first prepared from polyA-containing RNA isolated from day-old chick polysomes as previously described 171. Preparation of the fraction of cDNA which is complementary to the most abundant lens sequences was carried out by modification of the method of Hastie & Bishop [17] as follows. Seventy nanogram of lens cDNA was reacted with 3.5 ua of lens mRNA in 20 ~1 of formamide hybridisation &ffer (0.5 M NaCl. 25 mM HEPES. DH 6.8.0.5 mM EDTA. 50 mM EDTA, 50% (v/v) deibnised formamide) in a siliconised capillary tube to a Rot of 1.5~ 10-r (10 times the R,,tf of the most abundant class of sequences r71). The contents of the canillarv were flushed out into 230 ~1 of nuclease assay buffer (50 mM sodium acetate, pH 4.5, 2.8 mM ZnSO,, 0.1 M NaCl, 20 pg/ml denatured rat DNA) and 20 units of Sl nuclease added. The mixture was incubated for 1 h at 37°C and the reaction stopped by extraction with an equal volume of phenol : chloroform (1 : 1) 0.05 % 8hvdroxvquinoline. The phenol layer was re-extracted with iM ~1 H,O and the supematants combined. RNA was destroyed by treatment with 0.3 N NaOH for 1 h at 3PC. The solution was neutralised and cDNA collected by passage through a Sephadex G-50 column. The cDNA isolated was reacted again with lens mRNA and the procedure repeated. After two cycles of hybridisation the yield of abundant cDNA was about 30 % of the input cDNA. Alkaline sucrose gradients showed the abundant cDNA to be of similar length to a sample of
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75
the original cDNA (300-350 nucleotides). Abundant cDNA prepared in this way is lens-specific, as judged bv its hvbridisation to lens mRNA with aR,,ft of 2.58x I&’ mol-set/l and failure to hybridise to embryo body or smooth muscle mRNA at R,,t values as high as 1X 1o;Lmol-sec/l[7].
Hybridisation
reactions
Appropriate volumes of RNA and abundant cDNA solutions in sterile distilled water were mixed, lyophilised and dissolved in formamide hvbridisation buffer as above. The ratio of mRNA to-cDNA in these mixtures was 20: 1 or greater. Aliauots of the hvbridisation mixture (I-10~@I) were sealed in siliconised glass capillaries. The capillaries were heated at 65°C for 5 mm, then incubated at 43°C for various times. The contents of the capillaries were flushed out with 0.25 ml of nuclease assay buffer (50 mM sodium acetate pH 4.5, 2.8 mM ZnSO,, 0.1 M NaCl, 20 pg/ml denatured rat DNA) and frozen. The proportion of cDNA in hybrid was determined by addition of 20 units of Sl nuclease and calculating the proportion of acidsoluble radioactivity after incubation for 1 h at 37°C. In control experiments this amount of Sl nuclease was shown to be capable of completelv degrading the amount of cDNA used in the-presence of 1 mg/ml E. co/i RNA. Some 2-3% of dav-old lens cDNA and IO-1 1% of abundant cDNA was resistant to Sl nuclease digestion in the absence of complementary sequences (double-stranded sequences are conserved during the preparation of abundant cDNA). Values for percentage hybridisation in the presence of complementary RNA sequences were corrected to allow for this.
RESULTS Hybridisation reactions using total RNA
For most of the culture stages studied only a small amount of material was available and so in general only total cytoplasmic RNA was prepared from individual stages. It was therefore necessary to see if the vast excess of rRNA present affected the behaviour of the mRNA in cell-free translation and hybridisation experiments. We have shown that total lens polysomal RNA and polysomal mRNA selected by oligo(dT)cellulose chromatography are translated in the cell-free system to give very similar crystallin profiles [8]. Fig. 1 shows the result of hybridising abundant cDNA to (a) day-old lens polysomal mRNA, and (b) Exp Cell
Res 122 (1979)
76
Thomson
Table 1. Dosage
et rrl. oflens-spc)c(fic
sequences
Source of material
RNA sample
Rdt
Day-old chick lens Day-old chick lens S-day embryo NR S-day embryo PE 30-day NR cultures 42-day NR cultures 60-day PE cultures
Total polysomal RNA Polysomal mRNA Cytoplasmic mRNA Cytoplasmic mRNA Total cytoplasmic RNA Total cytoplasmic RNA Total cytoplasmic RNA
2.77x 2.58x IX 6.05x 8.56X I .89x 7.20x
Dosage 10-Z lo-’ 102” IO’ 10-l 10-l 10-l
0.93 I 0.00026” 0.00043 0.030 0.136 0.035
R,,t& values were calculated using a least-squares programme which provided a single component curve which was the best fit for the data points. In reactions where total RNA was used, it was assumed that I % of the input RNA was participating in the reaction. The values for the dosage of lens-specific sequences are calculated relative to the level of crystallin mRNA sequences (i.e. those which are complementary to abundant cDNA) present in day-old chick lens polysomal mRNA, which has been given the value 1. 0 These values are estimated, as the hybridisations with S-day embryo NR mRNA did not reach saturation levels even at the highest R,,t values available.
day-old lens total RNA in conditions of mRNA excess. As 1% of the total RNA was selected as mRNA by oligo(dT)cellulose chromatography the Rot values for the total RNA reaction are calculated on the basis that 1% of the input RNA is participating in the reaction. It is clear that the
presence of rRNA has little or no effect on the hybridisation reaction. R,,tl values were calculated from both sets of data points using a least squares programme which provided the best fit for a single component curve [17] and these are shown in table 1. The values are very similar and cer-
7
1. Abscissa: log Rot; ordinate: % hybridisation. O-0, mRNA; m-M, total RNA. Day-010 ChicK lens polysomal mRNA (selected by two cycles of oligo(dT)-cellulose chromatography) or total polysomal RNA hybridised to abundant cDNA to various R,r values.
trll
Cell
KC\
122 (IY7Y)
23
30
35
42
2. Abscissa: time in cell culture (days); ordinate: identification and mol. wt of products which comigrate with crystallin subunits. Ten pg of total cytoplasmic RNA from NR cultures at various stages of development were translated in the cell-free system and the products of translation detected as described. Approximately equal amounts of radioactivity were added to each slot of the gel. The fluorograph was developed after a time sufficient to detect only the most prominent products.
Fig.
Fig.
12 19
Lens mRNA
3. Abscissa: log R,r: ordinate: % hvbridised. (Z&) O-O, Day-old chick lens polysom; mRNA; V-V. total cvtonlasmic RNA from 42-dav cultures of 8-day embryonic-NR; X-X, total cytopiasmic RNA from 30-day cultures of 8-day embryonic NR; W.-W, l -a total cytoplasmic mRNA from 8-day embryonic NR (data from two experiments shown). (Bottom) B-H, Total cytoplasmic RNA from 23-day cultures of 8-day embryonic NR; O---O, total cytoplasmic RNA from 19-day cultures of 8-day embryonic NR. RNA samples from day-old chick lens, 8-day embryonic NR or from culture material were hybridised to abundant cDNA to various R,t values. The solid lines are not computer fits to the data. The curves for lens and embryonic NR are shown for comparison. Fig.
tainly within the errors inherent in the experiment. Since abundant cDNA hybridises to day-old chick lens mRNA with kinetics very similar to those shown by the fastest hybridising component in an experiment in which total cDNA from day-old chick lens was used [7], it may be used to probe for lens specific sequences in total RNA preparations.
in cultured cells
11
Crystallin mRNAs in NR cultures We have shown that translatable crystallin mRNA is present in large amounts in 42 day cultures of g-day chick embryo NR [8]. In order to follow the appearance of crystallinspecific mRNA during the course of these cultures, we have prepared total cytoplasmic RNA from cultures harvested at various stages of development. The products of cell-free translation of the various RNAs were analysed on an SDS-polyacrylamide gel and a fluorograph of the labelled products is shown in fig. 2. In the early stages of the culture only a few proteins are synthesised in detectable amounts and none of these co-migrate with crystallins. By 19 days in culture proteins comigrating with crystallins, particularly ycrystallin and a P-crystallin with mol. wt 34000 can be detected and they gradually become more prominent at subsequent culture stages, so that by 42 days they represent most of the detectable products. We have shown previously that traces of both crystallins [4] and lens-specific mRNA [7] are present in freshly excised g-day embryo NR. Since translatable crystallin mRNAs become more prominent as the cultures progress, it seems likely that lens-specific mRNA as measured by hybridisation to abundant cDNA, should also increase. This is shown in tig. 3. During the early stages of the culture no hybridisation to abundant cDNA could be detected at a Rot up to 1X IO’. At 19 and 23 days in culture some lens-specific sequences were detected but only at very low concentrations. Some cDNA remained single-stranded even at the highest Rot values achievable in these experiments. (The Rot curve of RNA from freshly excised NR was constructed using data from an experiment in which mRNA was used, and so greater R,t values were achieved.) It is clear that most of the inExp CdRes
122(1979)
12 0
-
/
6
P
1 alImB -
=)
d.
Fig. 5. Abscissu: slice no.; ordinclre: radioactivity (cpm). A sample of the cell-free translation products of cytoplasmic RNA from 60-day PE cultures was immunoprecipitated with anti-total crystallin antiserum and analysed on an SDS gel as described previously [8]. The gel was sliced into 1 mm slices and the radioactivity in each determined. The migration of marker crystallins in a parallel slot of the gel is also shown.
when compared with freshly excised embryonic NR, and by 42 days a further 5-fold increase is seen (table 1). By this terminal stage the levels of these lens-specific sequences have reached approx. 10% of the levels found in day-old chick lens polysomal mRNA.
Cvystallins and crystallin mRNAs in PE cultures Fig. 4. An SDS-polyacrylamide gel was run as de- PE cultures are slower to develop than NR scribed in Materials and Methods. (a), (h) Stained protein bands; (c), (d) fluorograph of dried gel. (a) cultures and fewer lentoids develop in the Soluble proteins from 42-day cultures of 8-day em- terminal stages and so it would be expected bryonic NR; (b) soluble proteins from 60-day cultures that less crystallin would accumulate during of I-day embryonic PE; (c) cell-free translation prodthe course of the culture. A comparison of ucts of total cytoplasmic RNA from 42-day cultures of 8-day embryonic NR; (d) cell-free translation prod- the soluble proteins from the terminal ucts of total cytoplasmic RNA from 60-day cultures of stages of embryonic NR and PE cultures on 8-day embryonic PE. The migration of the major chick crystallin subunits in a parallel gel is shown for com- an SDS-acrylamide gel (fig. 4) shows that parison. whereas large amounts of crystallin have accumulated in the NR culture, proteins cocrease in lens-specific mRNA (relative to migrating with identifiable crystallins are total mRNA) is found during the later less prominent in the soluble protein from stages of the culture. A comparison of the the PE culture. Furthermore large amounts Rot* values shows that by 30 days in cul- of crystallin products are seen when mRNA ture lens-specific mRNAs have increased from 42-day NR cultures is translated in the relative to total mRNA by approx. lOO-fold cell-free system. Translation of mRNA tlvp
(‘r/l
Ke.s 122 (1979)
. rf .
Lens mRNA
l
.
A--
.
.
.
.
.
.
.
/
.
.
.
.
/
!:
. . .
.
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.
/
.
.1
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.
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,
2
3
logR,t; ordinate: % hybridised. RNA samples from day-old chick lens, &day embryonic PE or culture material were hybridised to abundant cDNA to various R,r values. The solid lines are not computer fits to the data. O-O, Day-old chick lens polysomal mRNA; V-V, total cytoplasmic RNA from 60-day cultures of I-day embryonic PE; W-W, total cytoplasmic mRNA from 8-day embryonic PE.
Fig. 6. Abscissa:
from 60-day PE cultures gave a number of products, few of which co-migrate with crystallins (fig. 4). Previously, we have confirmed that a large proportion of the products of cell-free translation of 42-day NR culture mRNA were crystallins, by immunoprecipitations with anticrystallin antibodies [8]. Fig. 5 shows the result if a similar experiment using cell-free translation products of RNA from 60-day PE cultures. Only a very small proportion of the TCAprecipitable radioactivity was precipitated using antitotal chick crystallin antiserum (6 %, as opposed to 4-5 % in control experiments using non-immune serum). When run on an SDS-acrylamide gel a proportion of the radioactivity in the immunoprecipitate co-migrated with crystallin subtlnits. Some radioactivity is also seen in the high molecular weight region of the gel. The identity of this material is unclear but the most likely possibility is that these are lens membrane proteins, since lens membrane components run in this portion of the gel and the antiserum used has some specificity towards 6-791810
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cells
79
membrane proteins (Odeigah & Clayton, unpublished). However, by hybridisation to abundant cDNA, the amount of lens-specific mRNA present in 60-day PE cultures showed an 80-fold increase over the levels of these sequences present in freshly excised pigmented epithelium (fig. 6, table 1). Relative to total mRNA in each case, this is close to the level reached by NR cultures at 30 days of development. This is well short of the levels reached by NR cultures by the terminal stage and approx. 1% of the level of these sequences in day-old chick lens polysomal
mRNA.
DISCUSSION Whilst the cDNA complementary to the abundant mRNA sequences in the day-old lens has in this paper been referred to merely as lens-specific, there is now very good evidence that it is complementary only to crystallin mRNAs. This evidence can be summarised as follows: (1) The kinetic complexity of the cDNA suggests that it corresponds to only a small number (approx. 4) of different mRNAs [7]. (2) When day-old chick lens soluble proteins are separated on an SDS-acrylamide gel 7-9 prominent bands are seen and these are all identifiable crystallins [ 141. (3) Similarly, when day-old chick lens mRNA is translated in a cell-free system only crystallins are prominent products [8]. (4) The amounts of mRNA sequences complementary to the abundant cDNA increase markedly in two different cell culture systems where the amounts of both crystallins and translatable crystallin mRNAs are known to be increasing (this paper). Absolute proof of the nature of the abundant cDNA would require isolation and unambiguous identification of individual mRNAs after hybridisation to abunExp Cell Res 122 (1979)
dant cDNA, but any conclusion other than that the abundant cDNA is in fact crystallinspecific cDNA seems highly unlikely. The possibilities that (1) abundant mRNA _sequences may not include those coding for minor crystallins, and (2) cross-hybridisation between related but distinct mRNAs, may explain the discrepancy between the number of mRNA sequences represented in abundant lens cDNA and the number of crystallin protein subunits. In fact, hybridisation of 6-crystallin mRNA to its cDNA transcript occurs with kinetics which show a single sequence [19] while the protein consists of at least two similar but distinct subunits [ 14, 181. Under the conditions used here a single purified mRNA (globin mRNA) reacts with its cDNA copy over a range of 2.5 units on the log R,,t scale [20] and the lens mRNA-abundant cDNA reaction occurs over a similar range. If, in the heterologous reactions with RNA from NR and PE cultures, the relative concentrations of individual crystallin mRNAs were very different from those in day-old chick lens mRNA, the reaction would no longer occur as a single kinetic component. The hybridisation data from NR and PE cultures cannot be distinguished from that expected for a single component, but concentration differences of less than about ten-fold would not be detected. Since the plateau levels reached in the heterologous hybridisation reactions are similar to those reached in the homologous reactions then most or all of the mRNA sequences which are present in the day-old lens and complementary to crystallin cDNA are also present in the mRNA from the cultured material. In this paper two independent techniques show that crystallin-specific mRNA is increasing during culture of embryonic NR
and PE. It is important to correlate these results with the amounts of crystallin protein accumulated during the culture period. Using haemagglutination inhibition assays, de Pomerai et al. [4] have quantitated crystallin accumulation at various stages during culture of embryonic NR. Relative to the levels of crystallin mRNA or crystallin protein present in the day-old chick lens, the accumulation of crystallin specific mRNA and protein show similar patterns. A large increase occurs in both cases over the course of the culture period, mainly during the later stages. The most obvious interpretation of this data is that transdifferentiation in NR cultures is accompanied by transcription of crystallin genes to produce increasing amounts of crystallin mRNAs which are in turn translated. Crystallins accumulate until they account for a substantial proportion of the newly synthesised [8] and this paper) and accumulated [4] protein. We have not examined the levels of crystallin-specific mRNA in nuclear RNA, or the rate of mRNA breakdown, to see whether some post-transcriptional event could account for the increases seen in cytoplasmic mRNA. As both the number of cells and the amounts of crystallinspecific mRNA increase substantially during the culture it is unlikely that such a mechanism could contribute significantly to the overall sequence of events. Transcriptional control mechanisms have been proposed for other culture systems where differentiation of a particular cell type is occurring, e.g. for globin mRNA in cultured Friend erythroleukaemia cells [21]. In cultures of chick embryo muscle cells Paterson & Bishop [22] found no change in the total mRNA complexity during myogenesis (approx. 17000 sequences), but found large increases in the amounts of a small number of mRNAs. At the same time
Lens mRNA
there was increased synthesis of a small number of proteins when mRNA was isolated from the cultures and translated in a cell-free system. The total complexity of mRNA from lens cells is thought to be somewhat less [7] than that found in these muscle cells and in other chick tissues [23]. Perhaps this is because the lens consists of two cell types and is metabolically somewhat inactive. While a transcriptional control mechanism for crystallin gene expression in NR cultures is the most obvious interpretation of the data, other secondary levels of control are not excluded. There is good evidence that the accumulation of individual crystallins in NR and lens epithelium cultures is independently regulated [4]. A suitable assay for individual mRNAs would be required to determine whether this noncoordinate regulation is reflected at the mRNA level. The possibility that individual mRNAs are translated in vitro at differing efficiencies precludes the use of heterologous cell-free translation for such an analysis. However, DNA probes complementary to individual mRNA species provide a means to study these questions. Experiments to this end are currently in progress. We are grateful to Ross Poultry Ltd., Newbridge, Midlothian, for the supply of day-old chicks and fertile eggs. We thank Christine Wilkinson for skilled technical assistance and Drs R. Williamson and D. E. S. Truman for discussions. The work was supported by grants from the MRC and the Cancer Research Campaign. J. F. J. was supported by a postdoctoral research fellowship from Fight for Sight, Inc., New York City.
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81
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Okada, T S, Itoh, Y, Watanabe, K & Eguchi, G, Dev biol45 (1975) 321. de Pomerai, D I, Pritchard, D J & Clayton, R M, Dev biol60 (1977) 416. Clayton, R M, Campbell, J C & Truman, D E S, Exp eye res 7 (1%8) Il. Bours, J & van Doorenmaalen, W J, Exp eye res 13 (1972) 236. Jackson, J F, Clayton, R M, Williamson, R, Thomson, I, Truman, D E S & de Pomerai, D I, Dev biol65 (1978) 383. Thomson, I, Wilkinson, C E, Jackson, J F, de Pomerai, D I, Clayton, R M, Truman, D E S & Williamson, R, Dev bio165 (1978) 372. Harris, S E, Rosen, J M, Means, A R & O’Malley, B W, Biochemistry 14 (1975) 2072. Ross, J, Gielen, J, Packman, S I, Kawa, Y & Leder, P, J mol biol87 (1974) 697. Suzuki, Y & Suzuki, E, J mol biol88 (1974) 393. Pelham, H R B & Jackson, R J, Eur j biochem 67 (1976) 247. Laemmli, U K, Nature 227 (1970) 680. Thomson, I, Wilkinson, C E, Bums, A T H, Truman, D E S & Clayton, R M, Exp eye res 26 (1978) 351. Bonner, W M & Laskey, R A, Eur j biochem 46 (1974) 83. Laskey, R A & Mills, A 0, Eurj biochem 56 (1975) 335.
17. Hastie, N D & Bishop, J 0, Cell 9 (1976) 761. 18. Reszelbach, R, Shinohara, T S & Piatigorsky, J, Exp eye res 25 (1977) 583. 19. Zelenka, P & Piatigorsky, J, Exp eye res 22 (1976) 115. 20. Young, B D, Harrison, P R, Gilmour, S, Bimie, G D, Hell, Ai Humphries, S & Paul, J, J mol biol 84(1974)555.
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Received August 3 1, 1978 Revised version .__ . received .- .--- March 12, 1979 Accepted March 15, 1979
Exp Cd/
Re.\ 122 (1979)
