Characteristics of DNA from the oyster, Crassostrea gigas

Biochimica et Biophysica Acta, 335 (1973) 35-41

Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97888

C H A R A C T E R I S T I C S OF D N A F R O M T H E OYSTER, C R A S S O S T R E A

GIGAS

KIRK W. McLEAN* and ARTHUR H. WHITELEY Department of Zoology and the Friday Harbor Laboratories, University of Washington, Seattle, Wash. 98195 (U.S.A.)

(Received July 24th, 1973)

SUMMARY D N A was isolated from sperm of the oyster, Crassostrea gioas, and characterized as to guanine--cytosine content, presence of satellite D N A , and renaturation kinetics. The value for G + C content obtained from the thermal denaturation profile (Tm ~-- 82.5 °C) was 32.2 ~ , that from the buoyant density (1.693 g/cm3), 33.6 ~o. These experiments also demonstrated the presence of ( G + C ) - e n r i c h e d regions in the D N A which appeared as a high-density satellite (57.1 ~ ) in CsC1 gradients. The renaturation kinetics revealed at least three different degrees of sequence repetition present in the oyster genome: unique genes (70 ~ of the total DNA), moderately repeated genes (23 ~ ) , and highly repeated genes (7 %). The adjusted Cot~ of the unique fraction was used to calculate a minimum genome size of 7.6 • 1011 daltons per haploid set of chromosomes.

INTRODUCTION There have been a number of recent studies on the role of gene expression in early development which have concentrated on embryos whose development is regulative [1 ]. In a regulative embryo, each of the early blastomeres is capable of developing into a whole embryo. In another type of development, mosaic, isolated blastomeres are incapable of full development, as the fate of the embryonic regions has already been determined in the egg. In contrast with regulative embryos, relatively little is known about the role of gene expression in controlling early development of mosaic embryos. We analysed certain aspects of R N A synthesis in the embryo of the oyster, Crassostrea gigas, as an approach to the problem of gene expression in mosaic development. As much of the work was based on nucleic acid hybridization, it was necessary to characterize the genome to determine what part was involved in the hyAbbreviations: Cot, concentration of DNA ×time (moles of nucleotide × s/l); Cotl/z, Cot value at which the reassociation reaction is half-completed; SSC, 0.15 M NaCI4).015 M sodium citrate (pH 7.2); Tin, midpoint of the thermal transition of DNA. * Present address: Department of Biochemistry, University of Washington, Seattle, Wash. 98195, U.S.A.

36 bridization experiments. In addition, characterization of the oyster genome allows a comparison with genomes of other animals in which gene expression has been studied. In this paper, we report some of the characteristics of the oyster genome as revealed by thermal denaturation, isopycnic centrifugation and renaturation kinetics, and compare these characteristics with those of other invertebrate genomes. MATERIALS AND METHODS

DNA extraction The method used for DNA extraction from Crassostrea sperm was based on that of Whiteley et al. [2, 3]. Sperm was lysed in EDTA-Tris buffer containing pronase B (Calbiochem) and sodium dodecylsulfate and incubated for 16 h at 37 :C. Protein was removed by shaking with chloroform-octanol (10:1, v/v) and the DNA precipitated with ethanol. Additional purification steps, derived from the Marmur procedure [4], were done on the D N A used for isopycnic centrifugation and for renaturation analysis. The DNA was dissolved in 1 x S S C (0.15 M NaC1-0.015 M sodium citrate (pH 7.2)) and digested with heat-treated ribonuclease (Worthington) at 37 °C for 30 min. Pronase B (25/~g/ml) was added and incubation continued for 3 h. The solution was shaken repeatedly with chloroform-octanol until no interphase pad was present. The D N A was precipitated, dissolved in 0.1 × SSC, 1 ml of 3.0 M sodium acetate-1 mM E D T A (pH 7.0) added per 9 ml solution, and the DNA precipitated with 0.54 vol. isopropanol. The D N A was stored cold in 0. I × SSC. Thermal denaturation The temperature of a D N A sample in 1 x SSC in a stoppered cuvette (1-cm light path) was steadily increased at 15 °C per h. Both temperature and change in Az6o ,m were continuously recorded with a Gilford 2000 spectrophotometer. Buoyant density Buoyant density of the D N A was determined by equilibrium centrifugation in 8 molal CsCI-0.02 M Tris (pH 8.5), p = 1.715 g/cm 3. Centrifugation was in a Beckman Model E analytical ultracentrifuge equipped with a photoscanner, multiplexer, and monochromator, using an An-F rotor with double-sectored cells. Equilibrium was reached after 19 h at 40 000 rev./min. Renaturation Purified DNA was sheared at 12 000 lb/inch 2 in a French pressure cell with a ball valve, followed by sonication. The D N A fragments were concentrated, and filtered through Sephadex G-50 coarse (Pharmacia). The center fractions of the excluded peak were pooled to give fragments of about 430 nucleotide pairs [5]. Samples were diluted to 630-1000/~g/ml with phosphate buffer and 40 ~o formamide (Eastman) [6]. The samples were placed in stoppered cuvettes (l-mm light path, Precision) in a Gilford 2000 spectrophotometer and the DNA denatured by increasing the temperature to 80°C for 10 min. The temperature was reduced to 37°C and renaturation monitored by continuously recording the A27 o ,m" The value for total hyperchromicity was corrected for decrease during cooling due to single-stranded collapse by considering the A270 ,m value at which the temperature reached the Tm (53.7 °C) during the cooling

37

phase to represent 100 ~o denatured D N A [7]. Renaturation was also considered to begin at this time. Cot values were calculated using the value Cot 1 = 83 pg/ml of D N A incubated for 1 h [8]. The percent of the D N A remaining denatured at different Cot values was plotted against log Cot. RESULTS

Thermal denaturation The hyperchromic shift of Crassostrea D N A due to thermal denaturation was 39 ~o and the T~ was 82.5 °C (data not shown). The G + C content for the D N A as calculated [9] from the Tm is 32.2 ~o- The thermal denaturation data can be used to reveal heterogeneous regions in the D N A by plotting the percent increase in absorbance against temperature on normal probability paper [10]. A random distribution of base pairs within the D N A results in a linear plot, whereas a concentration of any base pair within a region ~ill produce a change in slope. Fig. 1 is such a plot of D N A denaturation data for Crassostrea. 75 ~ of the D N A has an absorbance shift with a Tm of 82 °C while 25 ~ has a Tm of 86.6 °C. The G + C contents calculated for these two fractions are 31 and 42 o/ /O~ respectively. 99989590-

5so-

~ 70~ 60-

; 5o~ ~m~20105" 2. 1. 0

78

go

~2

~4

~6

Temperature *C

~8

Fig. 1. Normal probability plot of a thermal denaturation curve of C. gigas DNA. The percent of total change in absorbance occuring by each temperature was calculated and plotted against temperature on normal probability paper. The arrows mark, from left to right, the Tm (82 °C) of the major

fraction, the mean Tin, and the Tm (86.6 °C) of the minor portion.

Buoyant density The Crassostrea sperm D N A was also characterized by isopycnic centrifugation. Fig. 2A shows a n .4265 nrn scan of oyster D N A at equilibrium. Micrococcus lysodeikticus D N A (O = 1.731 g/cm 3) was used as a reference marker. The buoyant density of the oyster D N A was calculated to be 1.693 g/cm 3. The G q - C content calcu-

38 lated from the buoyant density [11] is 33.6 ~ , which is in good agreement with the value of 32.3 ~ determined from the Tm. Fig. 2B shows the results of an equilibrium centrifugation in which the main band was intentionally overloaded. A satellite peak sedimenting farther than the main band appears when the gradient is scanned at 265 nm, but disappears at 300 rim, indicating that it represents DNA. Using the main band as a reference, the buoyant density of this satellite is 1.716 g/cm 3, giving a G + C estimate of 57.1 o/. This satellite would account for part of the high-melting region seen in the probability plot. 1.2E

1.0-

~ 0.8~ 0.6-

< 0.40.2-

i

1.693 g / c m 3

C. gigas

J

i

1.731 g / c m 3

M. lysodeikticus

!

f,l 1.693g/cm 3

1.716 g/cm 3

Fig. 2. CsC] equilibrium centrifugation of Crassostrea DINA. D N A was centrifuged to equilibrium (40 000 rev./min, 22 h) in a Beckman Model E analytical ultracentrifuge, using an An-F double-

sectored rotor. The centripetal end of the cell is to the left in the figures. (A) Tracing of a 265-nm optical scan of a mixture of 0.72/~g of Crassostrea DNA and 0.3/~g of M. lysodeikticus DNA centrifuged to equilibrium. Density of Micrococcus DNA is 1.731 g/cm3, density of Crassostrea DNA is 1.693 g/cm3. (B) Tracing of 265 nm (-) and 300 nm (- - -) optical scans of 3.2/~g of Crassostrea DNA centrifuged to equilibrium. Main band density is 1.693 g/cm3, satellite density is 1.716 g/cm3.

Renaturation kinetics

The oyster genome was also characterized by the kinetics of D N A renaturation. Analysis of the rate of reassociation of D N A under controlled conditions provides two useful sets of information: the relative proportions of repeated and unique nucleotide sequences present in the genome, and the size and complexity of the genome [8]. The relative proportions of repeated and unique sequences in the genome can be estimated from the changes in reassociation rate found from the renaturation curve. The size of the genome being investigated and its genetic complexity can be estimated by comparing its rate of reassociation with that of a genome of known size. If the salt concentration, temperature and D N A fragment size are held constant, the reassociation rate depends on the product of the concentration of D N A × incubation time, or Cot. The renaturation kinetics can be determined by plotting the percent denatured D N A against log Cot. Such a Cot curve for Crassostrea D N A is given in Fig. 3. Renaturation data for Bacillus subtilis D N A obtained under identical conditions are also plotted for comparison (Mizuno, S., personal communication).

39 100

i

20

i

i

B, subtilis

10

3.8

* 00"1

D

100

~

,

400 1200

,'%...

,

,

101' '2~ 103 Cok ( moles nucleotide.s "1. I"1

104

Fig. 3. Renaturation kinetics for Crassostrea DNA. D N A was sheared at 12 000 lb/inch 2, sonicated, and concentrated. It was brought to a concentration of 630-1000/~g/ml in phosphate buffer and 40 % formamide and placed under mineral oil in 1-mm light-path, stoppered cuvettes. The temperature was held at 80 °C for 10 min to denature the DNA, then dropped to the incubation temperature of 37 °C. Renaturation was considered to begin after the temperature had reached the Tm during the cooling phase. The /1270 nm was continuously recorded in a Gilford 2000 spectrophotometer. The percent of the DNA remaining denatured was calculated from the .427onm and plotted against the log Cot.

There are three regions of different frequency shown on this graph: a rapidly annealing component which renatures by Cot 2 (7 % of the DNA); an intermediate component, renaturing between Cot 2 and Cot 80 (23 %); and a slowly reassociating fraction, Cot greater than 80 (70 %00)-Originally, the designation of parts of such a curve as representing reiterated or unique genes was done for calf DNA in which there is a plateau of almost two Cot decades between the two fractions [8]. Although such a designation for the parts of a more gradual curve such as found for the oyster is more arbitrary, nevertheless it is probable that the fast, intermediate, and slowly annealing fractions of the oyster genome represent highly repeated, moderately repeated, and unique gene sequences. This conclusion is supported by the fact that the slowly renaturing fraction anneals more slowly than 95 % of the B. subtilis DNA, a characteristic of unique sequences in eukaryotic genomes [8]. A distinctive feature of the renaturation kinetics of any given genome is the Cot value at which the reaction is half completed, or Cot~. [8] .With the conditions under which the data in Fig. 3 were obtained, B. subtilis DNA has a Cot~r of 3.8 while Crassostrea DNA has a Cot~ of 400, indicating the much greater complexity of the latter genome. The actual degree of complexity must be calculated using the Cot~ of the unique sequences, rather than of the whole genome, as the linear relationship between Cot, and sequence diversity holds only for non-repeated sequences [7,12]. The Cot~r for the slowly renaturing fraction is 1200 (Fig. 3) but this value must be corrected by the fraction of the unique sequences in the whole genome, as the rapidly annealing sequences increase the apparent Cot~r by diluting the unique sequences [7]. The corrected value is thus 1200 ×0.7 = 840. The Crassostrea genome is 840/3.8, or 221 times as complex as that of B. subtilis. Although the repetitive fraction does not contribute significantly to the sequence diversity, it does contribute to the overall size

40

of the genome, and therefore the oyster genome is 1200/3.8 or 316 times as large as the B. subtilis genome. Using a value of 2.4- 109 daltons as the size of the B. std~tilis genome [13], the oyster genome is calculated to be 2.4 • 109 daltons ×316 7.6. 10 ~ daltons. DISCUSSION

The G + C content of DNA from Crassostrea (32.2-33.6 o/ jo) is somewhat lower than that found for DNA from many other marine invertebrates. For example, the G-f-C content of DNA from the limpet Acmaea scutum is 41 ~ [14]; from the gastropod llyanassa, is 42 ~o [12]; and from the ascidian Ascidia callosa, is 40 ~ [15]. However, the G + C content of DNA from the bivalve mollusc Mytilus californianus is identical to that of Crassostrea (Gordon, W., personal communication). (G--C)-enriched satellites similar to that found in the oyster have been found in the genonaes of a number of invertebrates: the ascidian Ciona intestinalis [15]; the sea urchin Lytechinus variegatus [16]; the sand dollar Dendraster excentricus (Mizuno, S., personal communication); and the dipteran Drosophila hydei [7]. The high G + C satellite (63 %~,G - C) in Lytechinus has been shown to be composed mostly of ribosomal cistrons [I 6]. The buoyant density of the oyster satellite is slightly lower than that of the urchin (1.716 vs 1.722 g/cn'l 3) corresponding to a G ÷ C content of 57 ~o. If the oyster satellite does represent ribosomal cistrons, the lower density may indicate a lower G-t-C content for oyster rRNA. It was estimated from the Cot curve that about 70 ~o of the oyster genome consists of unique nucleotide sequences, 23 ~ of moderately repeated sequences, and 7 ~o of highly repeated sequences. These estimates are comparable to those found from similar curves for other invertebrates. Unique sequences form 60-80 ~ of the genome in Ciona [15], Dendraster [5], the sea urchin Strongylocentrotus purpuratus (Whiteley, A. H., unpublished), and D. hydei embryonic DNA [7]. Determination of these percentages from a renaturation curve of whole DNA may tend to overestimate the amount of unique genes present [12]. Therefore, the estimate that the repeated sequences form 30 ~ of the oyster genome is a minimal estimate. The size of the oyster genome, about 7.6 • 1011 daltons per haploid set of chromosomes, is similar to the size reported for the mussel Mytilus edulis, 1.86. 1012 daltons per diploid set, and to that reported for the clam Spisula solidissima, 1.34" 10 ~2 daltons per diploid set [17]. Such a genome is relatively large, being about half that of the calf [8] and of llyanassa [12], about ten times larger than that of Ciona [15] and about six times that of D. hydei [7]. ACKNOWLEDG EM ENTS

We would like to thank Dr Robert Fernald for making available the facilities at the Friday Harbor Laboratories, Dr Shigeki Mizuno for the B. subtilis renaturation calibrations, and Dr Charles Laird for the gift of M. lysodeikticus DNA. This work was supported by an N.D.E.A. Title IV fellowship, by P.H.S. training grant No. HD00266 from N.I.C.H.H.D., and by a grant from N.S.F.

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