Relative transcription from DNA of different degrees of redundancy in developing frog embryos

Copyright A// rights

Q 1973 by Academic Press, Inc. in any form resrrced

of reproduction

Experimental Cell Research 76 (1973) 289-296

RELATIVE

TRANSCRIPTION

OF REDUNDANCY

FROM

DNA

IN DEVELOPING

R. A. FLICKINGER,

J. C. DANIEL

OF DIFFERENT FROG

DEGREES

EMBRYOS

and R. A. MITCHELL

Department of Biology, State Univesity of New York at Buffalo, Buffalo, N.Y. 14214, USA

SUMMARY Frog DNA was separated into highly repetitious, moderately repetitious and least repetitious fractions which were hybridized to labeled RNA of isolated nuclei and whole cells of developing frog embryos and adult frog liver. There was a decrease in the number of kinds of D-RNA transcribed by all three classes of DNA in the nuclei during development, but an increase in the number of kinds of D-RNA in whole embryos during development. The thermal stability of heterologous hybrids of mouse and frog DNA indicate that it is the more repetitious sequences which are more conservative. The speculation is advanced that the general transcriptional pattern during development is based on the redundancy of the genome and the time of acquisition of genes during evolution.

In the present study we have separated Rana pipiens DNA into fractions of different average reiteration frequencies and hybridized these fractions with labeled RNA obtained from nuclei of frog embryos and adult liver to examine the qualitative transcriptive activity of each of these DNA fractions. Labeled RNA from whole embryos and livers was hybridized to DNA of different reiteration frequencies to assess the relative accumulation of different kinds of RNA in the whoZe cells. In order to learn which class of DNA is evolutionarily most conservative the sequence homologies of highly reiterated, moderately reiterated and least reiterated DNA of mice and frogs were compared.

Preparation of nuclei and RNA Nuclei from neurulae (stage 14 of Shumway [16]) and swimming larvae (stage 25) and adult frog livers were prepared according to Daniel & Flickinger [7]. RNA was prepared from the various sources using a stepwise hot phenol extraction procedure [12]. The RNA was then methylated in vitro with carrier-free dimethyl-3H-sulfate [17], followd by an additional DNase treatment. a pronase treatment (500 .&ml in 0.13 M Tris-HCl, pH 8.6 for 90 min at 37°C) and two additional phenol extractions. The purified preparations were then passed through Sephadex C-25 in 0.15 M NaCl to remove small molecular weight contaminants. then loaded on a hvdroxvaoatite column held at 80°C and eluted with 0.2 ‘M phosphate buffer (PB), pH 6.7. The RNA was exhaustively dialysed against 0.15 M NaCl, precipitated with 3 vol of ethanol and redissolved in the hybridization medium. Specific activities ranged from 8 000 to 23 000 dpm/pg. RNA purified in this manner gave very low background levels of radioactivity on blank filters which were 0.002 % of the input counts in the hybridization experiments.

Construction of frog Cot curves MATERIALS

AND

METHODS

The degrees of hybridization of labeled RNA of frog embryos and adult liver to DNA of each repetition frequency were determined in the following manner. 20-

721816

Frog DNA was isolated from erythrocytes [12] and E. coli DNA was purchased from Worthington Biochemical Co. DNA was dissolved in 0.01 M NaCl at 500-l 000 pg/ml and sheared at 40 000 p.s.i. or 50000 psi. by two passages through a French Exptl Cell Res 76 (1973)

290

R. A. Flickinger et al.

pressure cell (Aminco). C,r curves were constructed according to the methods of Britten & Kohne [3]. DNA for determination of the percentage reassociation was passed through hydroxyapatite at 60°C in 0.4 M phosphate buffer to remove crosslinked fragments. Samples were lyophilized, dissolved in distilled water, then dialyzed for 2436 h at room temperature into the desired phosphate buffer concentration. Phosuhate buffer. DH 6.7. was rendered free of divalent -metal cations’ by passage through Chelex 100 (BioRad. New York). Samnles (0.15-1.0 ml) were sealed in 1 ‘ml glass ampules and denatured bv heating at 98°C for 5 min. then transferred to a water bath and incubated at 60°C. Following incubation, samples were diluted to 0.12 M PB if necessary and applied to the column. Solutions were allowed to equilibrate for 2-3 min at 60°C before being adsorbed on the hydroxyapatite. Percent reassociation to each Cot point was calculated as the proportion of the total eluted DNA which was bound to hydroxyapatite in 0.12 M PB at 60°C.

Fractionation of DNA into repetition frequency fractions As a source of labeled frog DNA. 1000 tailbud embryos were cut into dorsal-axial and belly regions [II] and incubated in Niu-Twitty saline [14] containing W-thymidine (5 &i/ml) for 24 h, after which DNA was prepared. Frog red blood cell DNA was adjusted to a specific activity of 69-139 dpm/pg with the W-labeled DNA prior to shearing. Separation of the mixture of the W-labeled DNA plus unlabeled sheared DNA into arbitrary fractions of highly reiterated (HR), moderately reiterated (MR), and least reiterated (LR) DNA for RNA-DNA hvbridizations was achieved in the following way. The sheared DNA was denatured by heating at 98°C for 5 min. In order to separate the DNA into arbitrary fractions of different -degrees of redundancy, the sheared and denatured DNA was incubated at 60°C to a Cot (moles of nucleotides per 1x time in set) of 300, where the reassociation of single copy DNA begins to dominate the reaction, and then loaded on a hydroxyapatite column at 60°C. The least repetitious (LR) sequences were eluted as single-stranded DNA with 0.12 M phosphate buffer (PB) and collected. The bound fragments representing mainly reiterated sequences were eluted with 0.4 M PB, dialysed against 0.12 M PB, denatured and incubated to a Cot of 0.2 at which DNA sequences with a repetition frequency greater than about 1 000 were reassociated. At this COt the most highly repetitious (HR) sequences reassociated and were bound to the hydroxyapatite while the moderately repetitious (MR) DNA seauences were eluted with 0.12 M PB. The highly repe’titious DNA (HR) was finally eluted with 0.4 M PB. All the DNA fractions were dialysed against 0.015 M NaCI, lyophylized, and then dialysed against 0.01 x SSC (1 x SSC is 0.15 M NaCl, 0.015 M sodium citrate).

Hybridization

procedures

The three separated fractions of W-DNA were denatured (98°C for 10 min), quick cooled and Exptl Cell Res 76 (1973)

applied to nitrocellulose filters (Schleicher & Schuell B-6, 27 mm) in 8 x SSC essentially as described by Gillespie & Spiegelman [9]. Unsheared and denatured E. coli and frog DNA was trapped on filters with 6 x SSC. Hybridization was carried out in an incubation medium consisting of 1 M NaClO,, 50 % formamide plus 0.01 M Tris-HCl, pH 7.2, and 0.0025 M sodium versenate at 37°C. The use of formamide permitted the use of a low temperature which minimized nucleic acid degradation during long incubations [l]. The use of 1 M NaClO, increased the renaturation rate and permitted the use of shorter incubation times. For purposes of estimating equivalent C, t values, 1 M NaCIOa was assumed to increase the renaturation rate by lo-fold [15]. After incubation, the filters were washed in 1 M NaClO,, 50% formamide at 25°C for 30 min, treated with RNase A (200 pg/ml) in this same medium for 45 min, and washed again. Filters were counted for RNA (3H) and DNA (‘“C) according to the method of Daniel et al. 181. The validity of the method for estimating amounts of DNA remaining on the filters following incubation was tested in the following way. Large-numbers of control filters containing each of the three DNA repetition fractions were incubated in the same way as experimental filters, then assayed for DNA by counting or by the Burton diphenylamine reaction [5] (table I). It can be seen that W-DNA prepared from embryos was neither selectively lost nor retained during the incubations, compared to total DNA.

Comparison of degree of divergency in each DNA reiteration class using interspecific hybrids Tritium-labeled frog DNA was extracted from 300 tailbud explants incubated for 36 h in Niu-Twitty saline 1141 containing SH-thvmidine at 5 oCi/ml (19.5 Ci/mole). The spec. act-of DNA was 245 bO0 dpm/pg. The DNA was sheared at 40000 p.s.i. and fractionated into the three repetition fractions as already described. Mouse DNA was isolated from liver nuclei, sheared at 40 000 p.s.i. and mixed with each fraction of frog DNA at a mouse to frog ratio of 2 000: 1 in 1 M Dhosohate buffer containine 2.5 mM EDTA. The mjxtures were heated at 98°C for 15 min, then incubated at 65°C. Following the incubations, samples were frozen for later analysis. Samples were diluted to a concentration of 0.05 M phosphate buffer and adsorbed on hydroxyapatite at 50°C. Unreassociated material was washed from the column in 0.14 M PB. The temperature was raised in 34°C intervals while 0.14 M PB passed through the column. Fractions were collected and assayed for mouse DNA at A,,, and for frog 3H-DNA by collecting TCA precipitates on glass fiber filters and counting.

RESULTS Fractionation of D NA according to redundancy A C,t curve for frog red blood cell DNA is shown in fig. 1. Frog DNA sheared at 40 000

Trans’cription and DNA redundancy

29 1

Table 1. Amounts of sheared DNA retained by nitrocellulose filters at the end of the hybridization incubations

DNA fraction Highly repetitious Moderately repetitious Least repetitious

Total pg DNA/filter at start of incubation5 filters 1.8 3.0 4.4

Incubation time

fig DNA/filter at end of incubation based on W dpm

10 min 24 h 36 4 days 16 days

0.84 0.43 0.43 0.35 0.35

Results are the averages of 3 or 4 expts with

Percent DNA lost from filter during incubation

Average percent DNA lost from each filter during incubation determined by Burton diphenylamine reactions on 14 filters

53 86

54 86

92

91

S.D.

p.s.i. was separated into redundant DNA and least redundant fractions at C, t 300. Highly repetitious DNA was defined as those sequences of average repetition frequency greater than 103. Total redundant DNA was therefore fractionated at C,t 0.2 to yield a moderately repetitious fraction (42 % of the total DNA) and a highly repetitious fraction (20 % of the total DNA). Using a single-cut procedure, each repeti-

tion frequency fraction is 70-80 % pure. For HR DNA, this means that 70-80~ of the sequencesreassociate by C,t 0.2. By the same token, LR DNA contains most of the single-copy sequences of the frog genome and some of the moderately repetitious sequences.The MR fraction contains some HR DNA and some of the single-copy sequences. Samples from a single fractionation were used in the hybridization experiments, allowing the calculation of the percent hybridization on the basis of the total DNA and summation of these fractions to yield the total percentage hybridization for each RNA tested. Stability of nucleic acids during extended incubations

Fi,. I. Abscissa: Cot (M/I x set) in 0.12 M phosphate buffer at 60°C; ordinate: % DNA reassociated. Rana pipiens Azso C, t curves. Upper curve obtained using DNA sheared at 50000 p.s.i.; lower curve obtained using DNA sheared at 40 000 p.s.i. Points are in duplicate. Points were determined using DNA of 8 different concentrations ranging from 0.0094 to 8.5 mg/ml, and were staggered so that overlaps occurred. For Cot values above 1000, DNA was dissolved in 0.36 M phosphate buffer. All Cot values were adjusted to the reassociation rate in 0.12 M phosphate buffer using a table constructed by Britten & Smith [4].

RNA and DNA were not degraded during the extended incubations as indicated by sharp G-100 Sephadex elution profiles of both nucleic acids incubated under the same conditions as experimental samples. Acid precipitable counts of RNA before and after incubation were similar (87-90~ of total). DNA-RNA

hybridization

experiments

The levels of hybridization of the labeled RNAs were determined by incubating each Exptl CeN Res 76 (1973)

292

R. A. Flickinger et al.

Table 2. Hybridization of labeled RNA to highly repetitious (HR), moderately repetitious (MR) and least repetitious (LR) frog DNA fractions Percentage hybridization to each fraction Source of RNA

HR

MR

LR

Total % hybridizationa

2.4kO.3 1.3 10.3

48.OF 3.2 11.1 F2.3

46512.1 lO.O& 1.1

38.4k2.2 llSkl.4

1.710.2 1.1 kO.2 5.5 +0.7

3.6 +0.5 5.9+1.5 16.7kl.l

4.110.4 4.3 kO.3 6.5 +0.7

3.410.4 4.3 10.8 10.6kO.9

2.1 kO.6 1.6kO.2

3.9kO.7 3.1 i-o.1

4.8 +0.9 5.4+0.1

3.9 10.8 3.lkO.6

Embryo nuclei

Neurula nuclei (stage 14) Larval nuclei (stage 25) Whole embryos

Early gastrulae (stage 10) Early neurulae (stage 14) Swimming larvae (stage 25) Liver

Adult liver Adult liver nuclei

a Each hybridization percentage was multiplied by the fraction of DNA in its repetition class. The total percent hybridization is the sum of these fractions. For example, the total percentage hybridization of neurula nuclear RNA to frog DNA is 2.4x0.20+48.0x0.42+46.5 x0.38.

with filters containing highly repetitious DNA, moderately repetitious DNA and least repetitious DNA filters. The amount of DNA remaining on each filter after incubation was determined by monitoring the Cl4 counts, and the amount of remaining RNA was represented by the 3H counts. The RNA concentrations used for the filters with highly repetitious DNA were 600 ,ug/ml, while other RNA concentrations ranged from 4 to 14 mg/ml. The results of incubating the different RNA preparations with each of the three DNA fractions is shown in table 2. The RNA

Table

preparation

variety of D-RNA molecules in nucZei decreases with development, while a greater variety of D-RNA accumulates in intact tranembryos. This is true for D-RNA scribed from all three reiteration fractions of the DNA. The degree of transcription from highly reiterated DNA was quite low compared with transcription from the less reiterated DNA sequences. If only one strand of DNA is transcribed, it appears that virtually all the genes are being transcribed in neurula nuclei. Incubations with HR and MR filters were to a C,t calculated to be 10 times that necessary for saturation of

3. Comparison of thermal stabilities of frog-mouse DNA hybrids

DNA

Incubation to equivalent C, t a

Percent renatured

T, of reassociated DNA

Mouse HR-frog Mouse IR-frog Mouse LR-frog

10 0.01 10000 5 50 ooo 25

26 49 63 51 79 26

75 76.6 79 71.5 82 66.7

AT,

7.5

11.3

15.3

23.0

a Calculated using a table constructed by Britten & Smith [3] as C,t in 0.12 M PB, 60°C. b From the relationship derived by Laird et al, [13]. Exptl Cell Res 76 (1973)

Percent mismatchb

Transcription and DNA redundancy

293

3

i 50

60

I 70

, 80

,

go b.wd

I

Abscissa: temperature, “C; ordinate: % total DNA eluted. Fig. 2. Integral plot of melt of the least repetitious frog DNA-mouse DNA hybrids (0) and reassociated mouse DNA (0). -Frog least repetitious 3H-DNA (0.21 pg) was mixed with 40 000 p.s.i. sheared mouse DNA (432 pg) in a combined volume of 0.15 ml of 1 M PB containing 2.5 mM EDTA. The mixture was denatured and incubated at 65°C for 6.8 days to a C,,t (mouse) of 50 000. The reassociated material was isolated on hydroxyapatite at 50°C in 0.14 M PB, then melted in 3-4”C steps. DNA remaining on the column at 93°C was eluted with 0.4 M phosphate buffer (PB). Fig. 3. Conditions as in fig. 2 except that moderately reiterated frog %H-DNA was added. The incubation was for 1.4 days to a mouse DNA C,t of 10 000. Frog-mouse DNA hvbrids (0): . ,, mouse DNA hybhds (0). Fig. 4. Melt of frog highly repetitious 3H-DNAmouse DNA hybrids (0) and of reassociated sheared mouse DNA (0). Conditions as in fig. 2 except that the ratio of mouse to frog DNA was 1 000: 1. DNA was dissolved in 0.14 M PB and incubated to a mouse DNA Cot of 10.

Figs 24.

4

d

50

60

70

_

SO

the slowest kinetic component in each fraction. Incubation of LR DNA filters to a COt of 1.68 x IO5 was sufficient to saturate most sites when nuclear RNA was used since the value for neurula nuclear RNA approaches the theoretical maximum of 50% hybridization. The specificity of the hybridization procedure is indicated by the low counts on blank filters (O-IO cpm in the tritum channel) and by the low level of hybridization of

neurula nuclear RNA to E. coli DNA (0.26 %) carried out under the same conditions as the experiments reported in table 2. In this instance, filters containing 1 lug E. coli DNA were incubated to an estimated RNA C,,t of 1.2 x 104. Degree of divergency in each DNA reiteration class

The degree of relatedness between HR, MR frog DNA fractions and the DNA of a Exptl Cell Res 76 (1973)

294 R. A. Flickinger et al. distantly related chordate was determined. It was hoped that the thermal stability of the heterologous duplexes formed would either increase or decrease in relation to the apparent reiteration frequency of the frog DNA fractions. Accordingly, tritium-labeled HR, MR or LR frog DNA was incubated with whole sheared mouse DNA to the C,, t required for complete self-reaction of mouse sequences of comparable redundancy. Trace amounts of frog DNA were added to mouse DNA to minimize homologous interactions. Melting temperatures (table 3) indicate that the thermal stability of reassociated mouse DNA increases as more limited sequences react. At the same time, frog-mouse hybrids show decreasing thermal stability (figs 2, 3, 4), which is opposite the trend observed for whole frog DNA or when each of the fractions is incubated with whole frog DNA. These data indicate that the frequency of base-pair substitutions that has occurred since divergence from a common ancestor is greatest in the slowly reassociating fractions of the DNA and least in the fast fractions. Assuming that base-pair substitutions occur randomly throughout the DNA, it appears that the more highly reiterated DNA sequences are more conservative. DISCUSSION Much of the sheared DNA is lost from the filters within the first hour of incubation in the hybridizations. However, the critical point is that the determination of the radioactivity of the sheared DNA on the filters at the end of the incubation period indicates the exact amount of DNA present. This is verified by the DNA determinations made on a large number of filters containing each of the DNA fractions incubated for the same length of time and in the same manner as for the actual hybridization incubation Exptl Cell Res 76 (1973)

(table 1). The ratio of 3H-RNA to total DNA on the (as determined by W-radioactivity) filters at the end of the incubations allows the determination of the percent of the DNA hybridized. Hybridization of labeled RNA of isolated nuclei of developing frog embryos to highly repetitious, moderately repetitious and least repetitious DNA revealed that the lower the degree of redundancy of the DNA, the greater the restriction of kinds of D-RNA transcribed during development. As development proceeds fewer kinds of D-RNA are transcribed in the nuclei and the least reiterated DNA has the greatest reduction in variety of D-RNA molecules transcribed. It should be noted that the variety of molecules of DRNA transcribed from highly repetitious DNA appears to be quite low compared to that from the moderately repetitious and least repetitious DNA. The reason that highly repetitious DNA synthesized fewer kinds of D-RNA than moderately repetitious DNA may be because fewer kinds of DNA sequences are present in the highly repetitious fraction. It could be argued that since greater amounts of labeled RNA or longer times would be needed to saturate the least repetitious DNA, that our results for this DNA fraction are not true saturation levels. This is unlikely for the nuclear RNA since labeled neurula nuclear RNA hybridizes with 46.5 % of the least reiterated DNA. This is near the maximum level of hybridization of 50 % if only one strand of the DNA is transcribed. The real importance of the hybridization levels is that the relative transcriptive roles of the DNA of each repetition class can be compared between stages. Even if saturating amounts of labeled RNA were not present which is unlikely, the same nuclear RNA preparation of a given embryonic stage is hybridized to the three

Transcription and DNA redundancy classes of repetitious DNA and it is these relative values which are compared. The high hybridization values of nuclear RNA hybridized to moderately repetitious and least repetitious DNA were not obtained for labeled RNA of whole embryos and this is attributed to the dilution of D-RNA by the vast amounts of cytoplasmic ribosomal RNA. Therefore true saturation was not obtained for RNA obtained from whole embryos. The importance of the hybridization levels obtained using whole embryo RNA is that the relative accumulation of D-RNA transcribed from each repetition class of DNA can be compared at each stage. This is possible since no matter what the dilution by ribosomal RNA, the same RNA preparation of a given embryonic stage is hybridized to the three classes of repetitious DNA. Comparisons between stages can also be made since the vast quantity of maternal ribosomal RNA is similar. The accumulation of a larger variety of D-RNA in whole embryos as development proceeds, despite an accompanying restriction of transcription in nuclei, can be ascribed to the increase in number of cells in a similar mass of embryo cytoplasm [lo]. Because cross-reaction can occur between D-RNA molecules made from one repetitious sequence and other members of the same family of sequences, the hybridization levels for highly repetitious and moderately repetitious sequences may be over-estimated. For this reason and because the least repetitious DNA contains the greatest variety of base sequences, transcription from the least repetitious DNA is qualitatively greatest in all cases examined. The variety of sequences transcribed from highly reiterated sequences is the smallest in every instance. Southern [ 181has emphasized that after a certain degree of divergence that reiterated squences may be so heterogeneous that they would act as

295

single copy sequences in hybridization experiments. He states that more stringent conditions of reassociation would increase the proportion of repeated sequences in what might appear to be single copy DNA. If the process by which genes are inactivated during development is random, genes which are present in many copies would be more likely to escape restriction than less reiterated genes. The data of table 2, which represents species of RNA rather than number of transcripts from each class of DNA, clearly follows the trend expected: the least reiterated sequences are restricted more than moderately reiterated sequences, while highly reiterated sequences are least affected. Embryonic determination and differentiation after gastrulation appear to be characterized by a progressive restriction of gene activity in the nuclei. The more redundant and more conservative DNA sequences transcribe the D-RNAs present in many copies that accumulate early in development, while the DNA which is less conservative, but more diversified, transcribed D-RNAs present in fewer copies that accumulate later in development [12, 191. The melting experiments with the heterologous hybrids between mouse DNA and labeled highly repetitious, moderately repetitious and least repetitious frog DNA, suggest that the greatest divergency exists in the least repetitious DNA and the least in the highly repetitious DNA. Similar results have been found by Britten & Kohne [2] and Laird, McConaughy & McCarthy [13]. The extent of homology is a function of thermal stability since this measured the extent of mismatching of base sequences. The decreased thermal stability of the less reiterated DNA sequences suggests these sequences are least homologous in the mouse-frog comparisons. These data appear to support the speculation that to some extent the sequence of accumulaExptl Ceil Res 76 (1973)

296 R. A. Flickinger et al. tion of D-RNA molecules during embryonic development reflects the scale of redundancy of the DNA sequences, with D-RNAs transcribed from more redundant and conservative DNA sequences accumulating before those transcribed from the less redundant and conservative DNA sequences [12, 19, 201. This may be the manner in which the sequence of differentiation in ontogeny is related to the sequence of gene acquisition during phylogeny [lo]. It is possible that cell division in the early embryos increases the number of nuclei in a similar mass of embryo cytoplasm as the cell size decreases and allows the accumulation of D-RNA transcribed from less redundant and conservative genes that will account for later differentiations. However, if more reiterated DNA sequences account for determinations early in development and less reiterated DNA accounts for those in later developments, this presents a difficulty. How can a cell whose determination depends upon a sufficient number of cell divisions to accumulate D-RNA transcripts from less reiterated genes bypass the determination controlled by more reiterated genes? This can be explained if cells determined later in development alter or modify the synthetic patterns of cells having the potentiality for an earlier type of differentiation. An example of this would be noradrenaline which is an end-product characterizing cells of the sympathetic nervous system, but also a precursor to adrenaline which is an end-product of the adrenal medulla [6]. Both these cell types are derived from the neural crest and it is possible that differences in number of cell divisions may influence their differentiation. It is clear that the method of hybridization employed in this study is not ideal since much of the sheared DNA is lost from the filters during the course of the incubation. However, the decrease of hybridization of Exptl Cell Res 76 (1973)

the sheared DNA fractions with nuclear and whole embryo RNA with development (table 2) is similar to the results obtained with unsheared DNA [7, 121 and supports the validity of the data. The significance of the results rests primarily upon the relative differences of hybridization between the stages. Such comparisons allow judgments concerning the relative transcriptive role of DNA of varying degrees of reiteration in development of the frog embryo. This research was supported by a grant from the National Institute of Health.

REFERENCES 1. Bonner, J, Kung, G & Bekhor, I, Biochemistry 6 (1967) 3650. 2. Britten, R J & Kohne, D E, Carnegie inst yearbook 66 (1967) 78. 3. - Science 161 (1968) 529. 4. Britten, R J & Smith ,J, Carnegie inst yearbook 68 (1970) 378. Burton, K, Biochem j 62 (1956) 315. 2: Caston, J D, Dev biol 5 (1962) 468. 7. Daniel, J C & Flickinger, R A, Exptl cell res 64 (1971) 285. 8. Daniel, J C, Green, R F, Mitchell, R A & Flickinger, R A, Anal biochem 37 (1970) 330. 9. Gillespie, D & Spiegelman, S, J mol biol 12 (1965) 829. 10. Flickinger, R A, Dev. biol, suppl. 4 (1970) 12. 11. Flickinger, R A. Miygi, M. Moser. C R & Rollins. E, De;biol 15.(196?)-414. 12. Greene, R F & Flickinger, R A, Biochim biophys acta 217 (1970) 447. 13. Laird, C D, McConaghy, B L & McCarthy, B J, Nature 224 (1969) 149. 14. Niu, M C & Twitty, V C, Proc natl acad sci US 39 (1953) 985. 15. Seamy, D G & MacInnis, A J, Biochim biophys acta 209 (1970) 574. 16. Shumway, W, .Anat ret 78 (1956) 139. 17. Smith, K D, Armstrong, J L & McCarthy, B J, Biochim biophys acta 142 (1967) 323. 18. Southern, E M, Nature new biol 232 (1971) 82. 19. Whiteley, A H, McCarthy, B J & Whiteley, H R, Proc natl acad sci US 55 (1966) 519. 20. Whiteley, H R, McCarthy, B J & Whiteley, A H, Dev biol 21 (1970) 216.

Received May 15, 1972 Revised version received July 31, 1972