studies on photoreactivation in gametes and zygotes of the sand dollar, echinarachnius parma

Studies on Photoreactivation in Gametes and Zygotes of the Sand Dollar, Echinarachnius parma’

JOHN S. COOK AND ALVIN F. RIECK Department of Physiology and Biophysics, N e w Y o r k Universi ty School of Medicine, N e w Y o r k , N . Y.; Department of Physiology, Marquet te Universi ty School of Medicine, Milwaukee, Wiscons in; and t h e M o u n t Desert Island Biological Laboratory, Salsbury Cove, Maine

It is well established that the intervals between fertilization and the early cleav- ages in the zygotes of marine invertebrates can, be prolonged by treatment with ultraviolet (UV) radiation, and that this cleavage delay can be mitigated by post-UV illumination with light in the spectral range 0.3-0.5 u. This latter phenomenon, known generally as photoreactivation or photorecovery (see Jagger, ’58 for review and references) has been described by Blum et al. (’50, ’51) and Marshak (’49) for ATbacia and by Wells and Giese (’50) for the California sea urchin Strongylo- centrotus purpuratus. Although their several results are basically similar, they remain at variance in a few important respects; quite possibly these differences can be related in part to the different species studied.

The sand dollar Echinarachnius parma provides very favorable material for study- ing the photobiology of cell division. In this paper we shall describe some of the basic characteristics of UV-induced cleavage delay and photoreactivation in this species. Owing to the discrepancies in the earlier work, which shall be pointed out subsequently, we have found it necessary to repeat some of those observations. In addition, we shall present data on reciprocity (dose-rate independence) of the UV lesion, on the maximum extent of photoreactivation, and on the time-dependence of photoreactivation during the cleavage cycle.

Since cleavage is delayed but not stopped by UV radiation (except at very high doses where the eggs are lysed), the term “photoreactivation” is less descriptive than “photorecovery.” The former term is more generally accepted, however, and the phenomenon we have studied is very probably

an expression of the same fundamental biochemical process which underlies photoreactivation in all biological systems; con- sequently, we have used the term “photoreactivation” throughout this paper.

METHODS

Gametes were collected by injecting a few tenths cm3 of 0.5MKC1 into the oral region of a sand dollar which had been inverted over a finger bowl filled with sea water. Eggs to be irradiated were made up into suspensions sufficiently dilute that the cells formed a single layer on the bottom of the dish. Concentrated sperm was diluted 1:lOO in sea water prior to irradiation, and subsequently diluted further 1 : 200 for use in fertilization. The depth of the irradiated suspensions was about 2 mm; gametes were gently stirred during the UV treatment.

Irradiation was carried out with either ( 1 ) a low-pressure mercury arc (Engel- hard Industries) with its principal output at 254 mcl, or (2) a Hanovia Utility Model quartz lamp with an emission from 226 to 405 my, peaking between 254 and 257 mu. No qualitative differences in results were found with these two sources. Radiation was monitored with a Westinghouse zirco- nium phototube, sensitive to 200-315 my radiation, and equipped with a counter which integrated this radiant energy over the period of exposure (Blum, Kirby-Smith and Grady, ’41).

After UV-irradiation, the gametes were divided into several Stender dishes and fertilized. These dishes were wrapped in aluminum foil, and those to be kept in the dark were covered with wrapped tops.

~

1This investigation was supported in part by a Public Health Service research grant ((2-2644) frpm the National Cancer Institute, Public Health Service.

77

78 JOHN S. COOK AND ALVIN F. RIECK

Those to be exposed to photoreactivating illumination were covered with ordinary glass tops. All dishes were then placed on a sea table 7 cm below a bank of two Gen- eral Electric “Blacklights” (BL), emitting between 320 and 450 mv with a peak at 365 mw. The running water of the sea table maintained the temperature within about 1°C during a given experiment. At appropriate intervals after fertilization samples of the zygotes were taken, added to 5% formalin in sea water, and counted for cleavage under high dry magnification. A well-defined cleavage furrow constituted the end-point, and 100-200 eggs were counted for each point on the curve. In the experiments reported here, fertilization exceeded 98% in all dishes.

In the following description of the experiments, the term “irradiation” will denote exposure of the gametes or zygotes to ultraviolet radiation, and the term “illumination” will denote exposure to reactivating light (BL).

RESULTS

1. sperm, and zygote

In figure 1 are depicted the results of a run showing several of the principal char-

Test for photoreactivation in egg,

D W > a w -I 0

c Z W 0

W a a

acteristics of the system. Eggs were irradiated with 8 X lo4 ergs/cm2 UV at 254 mv. Immediately thereafter both irradiated and control eggs from the same female were fertilized with normal sperm. From the graph i t is apparent that as cleavage is increasingly delayed under the various conditions, the slope of the curve falls off concomitantly. For subsequent presen- tation of the data, we have selected the time to 50% cleavage (t,,) as the index of the curve. It must be emphasized, however, that complete cleavage curves were obtained for each run, and the run was dis- carded if less than 98% cleavage was at- tained.

Figure 1 shows clearly the effect of “Blacklight” alone on the zygotes. The tso for the controls under the BL was about 6 minutes longer than that for the dark controls. This delay, also observed by Wells and Giese, was apparent in all our experiments, but usually did not amount to more than about three minutes.

The photoreactivation phenomenon is apparent in curves C and D. At this UV dose, irradiated eggs in the dark do not reach the midpoint of first cleavage until 36 minutes after the dark controls, where-

7 5 -

5 0 -

25 -

-I

/:

I I

130 140

MINUTES AFTER F E R T I L IZ AT1 ON Fig. 1 Effect of ultraviolet radiation and Blacklight illumination on first cleavage. UV-

irradiation of eggs prior to fertilization; BL illumination started immediately after fertilization. A - unirradiated controls in dark; B - unirradiated controls in BL; C - UV-irradiated eggs in BL; D - UV-irradiated eggs in dark. Eggs from a single female, fertilized with sperm from a single normal male. UV = 8 X lo4 ergs/cmZ. Temp. = 16°C.

PHOTOREACTIVATION I N THE SAND DOLLAR 79

TABLE 1 Time to 50% first cleavage ( t 5 0 ) of zygotes after UV-irradiation of gametes and illumination

of gametes or zygotes. Low-pressure UV urc (254 m p )

Gamete irradiated

Delay over control

Pre- Post-

illumination illumination uv fertilization fertilization t50

Eggs - (T = 15'C) + + + + (T = 15.5°C) -

+ +

Sperm

-

minutes 93

122 102 104 102

minutes 0

29 9

11 9

96 107 135 160

0 11 39 64

Sperm (T = 16OC) - - - 92 0

- + 94 2 - 151 59 - + 115 23

- - + +

~~

UV on eggs = 8 x 104 ergs/cmz; UV on sperm.= 3.2 x lo* ergs/cmz. Illumination in excess of PR,,, in all experiments.

as UV-irradiated eggs under BL are delayed only 15 minutes beyond the dark controls.

Table 1, compiled from three representa- tive experiments, summarizes the systems in which photoreactivation is or is not found. In these experiments, photoreactivation was always maximal (see section 4 of Results, below). (1 ) Photoreactivation can be induced in irradiated eggs either before or after fertilization, and with about equal effectiveness in either case (top section of table 1). (2) Control sperm are more sensitive to BL illumination than are the eggs or zygotes, a difference which is probably attributable to the partial filtra- tion of this radiation by compounds in the egg cytoplasm. If UV-irradiated sperm are used to fertilize normal eggs, the sperm and zygotes being kept in the dark throughout, a marked cleavage delay is observed. If UV-irradiated sperm are exposed to BL prior to their being used to fertilize normal eggs, no photoreactivation is observed. On the contrary, UV-irradiated sperm seem even more sensitive to these wavelengths than normal sperm, and the effect of the two types of radiation in sequence is to produce very long cleavage delay (middle section of table 1). ( 3 ) If UV-irradiated sperm are used immediately to fertilize normal eggs, and the zygotes are then ex-

posed to longer wavelength illumination, photoreactivation is observed (bottom section of table 1) .

All of these results are consistent with the results of Blum and his co-workers with Arbacia, who showed that UV-irradiation produced cleavage delay when nuclear material was present, but that photoreactivation was demonstrable only when (egg) cytoplasmic material was present.

2. Tes t for thermal reactions associated with the UV-lesion

Giese et al. ('56) have reported that brief dark periods following short flashes of ultraviolet enhance the deleterious effects of the radiation on the protozoon Didinium nasutum, an observation which implies the intimate association of "dark" or thermal reactions with the primary photochemical step. We have looked for thermal reactions in the UV-induced cleavage delay following Echinarachnius sperm irradiation. The first type of experiment tried was treating the sperm with continuous irradiation at various dose-rates (intensities) while maintaining the total UV dose constant. Table 2 gives the results of an experiment at a dose of 2.85 X lo" ergs/cma (Hanovia lamp; dose-rate vaned by setting the lamp at various distances

80 JOHN S . COOK AND ALVIN F. RIECK

TABLE 2 Time to 50% first cleavage of zygotes in the dark

and under illumination after UV-irradiation

UV dose = 2.85 X 105 ergs/crn2 in all cases.

of sperm at different dose-rates. Total

tso tso Dark Light

minutes minutes

Dose-rate

Unirradiated control 92.5 92

Lowest (7.92 X lo2 ergs)

cm2-sec. - -~ ~~

Intermediate

126 109

(3.96 >< lo3 ergs) cm2-sec. 126 108 ____- -__-

Highest (1.425 X lo4 ergs)

cm2-sec. 125 108 -~ -

from the sperm suspension). Gametes from a single male were treated with this dose over an 18-fold range of dose-rates. Immediately after irradiation, the sperm were used to fertilize aliquots of eggs from a single female, one aliquot corresponding

n w

w 1 0

v) (3 (3 w lL 0

I- z w 0 U w a

5

to each dose rate being placed under vis- ible illumination and one aliquot being kept in the dark. It can be seen from the table that the cleavage delay was the same at all dose-rates, and that photoreactivation occurred to the same extent in all cases. Similar results were obtained in the same intensity range at doses of 2.0 X lo5 and 6.3 X lo5 ergs/cm2.

The second experimental approach to the question of thermal reactions was to expose sperm to UV at various intensities as before, but with the radiation being interrupted by dark periods such that all samples received the same total dose in the same period of time. The dose-rate was again adjusted by placing the Hanovia lamp at various distances from the sample to be irradiated. At the lowest dose-rate the radiation was continuous. A rotating disc with one or two 20" wedge-shaped aper- tures was attached to the lamp housing and served to interrupt the higher intensity UV flashes of 18.5 msecs. with dark periods of 148 or 314.8 msecs. The results of such an experiment, together with data

I I 1 I I I I

- I1 160 I20 140 160

TIME AFTER FERTILIZATION, MINUTES Fig. 2 Delay in first cleavage of zygotes after flashing UV-irradiation of sperm prior to

fertilization of normal eggs. Open circles - unirradiated controls; open triangles - continuous UV-irradiation; solid triangles - UV flashes of 18.5 msecs. alternating with dark periods of 314.8 msecs. Solid squares - UV flashes of 18.5 msecs. alternating with dark periods of 148 msecs. Total UV dose-2.85 X lo5 ergs/cm2 in all irradiated samples; intensity adjusted so that dose was delivered in 6 minutes in each case. Zygotes in dark throughout. Temp. = 15OC.

PHOTOREACTIVATION I N THE SAND DOLLAR 81

on unirradiated controls, are shown in fig- cleavage delay is independent of the dose- ure 2, where it can be seen that the dark rate. The delay is prolonged, however, as periods were without effect on cleavage the total dose is increased. This is true delay. Over the ranges studied there is no whether sperm or eggs are irradiated, as is evidence of thermal reactions associated evident from table 3.a This progressive with the production of the UV lesion. effect of increasing dose appears to rule

out a “single-hit” action of the radiation 3. Effect of total ultraviolet dose on the cell nucleus.

The data presented in the preceding section indicate that the extent of the

TABLE 3 Time to 50% first cleavage ( t so ) o f UV-irradiated

zygotes incubated in the dark following ultraviolet irradiation

t50 Material UV dose irradiated ergsjcm2

Sperm (T = 14°C) 0 108

1.58 x 104 141 3.16 X 104 149 7.32 x 104 160

4. Test for maximum photoreactivation In the various biological systems where

studies have been made on the maximum extent of photoreactivation attainable, it has been observed that recovery from the ultraviolet lesion is almost never complete (Jagger, ’58). The data presented in this section will show that, for the Echinarachnius system we have investigated, the maximum photoreactivation is again less than complete mitigation of the ultraviolet effect.

Eggs (T = 15°C) 0 87

2 x 104 96 4 x 104 108 8 X 104 117 ( PRmax ) (96)

~ _ _ _ - Sperm irradiated with polychromatic arc. eggs with

monochromatic arc. Note different tempGratures of the two experiments.

W (3

> W

a a

I L m $ w ot- 0 3

140

130

120

I10

zThe two experiments summarized in table 3 are not comparable; they were made with different ani- mals at different temperatures and, most important, they utilized different ultraviolet arcs. Nevertheless, the indication that sperm are more sensitive to UV radiation than are eggs has been borne out in nu- merous experiments with the low pressure arc on the two systems. No effort has been made to c0mpa.m the relative sensitivities of the gametes on a quantitatlve basis; the sperm nucleus obviously lacks the pro- tection afforded the egg nucleus by an ultravlolet absorbing cytoplasm.

I I I I I I I I I

10 20 30 40 5 0 60 7 0 80

DURATION OF BLACKLIGHT ILLUMINATION, MINUTES

Fig. 3 Time to 50% first cleavage after UV-irradiation of eggs (8 x lo4 ergs/cm2 prior to fertilization) as a function of duration of BL illumination. Illumination started immediately after fertilization. Dashed line connects values for unirradiated controls kept in the dark (open circle, left) and in the BL (open circle, right). Temp. = 14°C.

82 JOHN S . COOK AND ALVIN F. RIECK

Our standard system consisted of a UV dose of 8 X lo4 ergs/cm2 given to unfertilized eggs. These eggs were then fertilized with normal sperm, and aliquots of the zygotes were treated with reactivating illumination of varying durations or at various periods between fertilization and first cleavage. In the first series of experiments with this system, the irradiated zygotes were illuminated for various durations of time, all starting immediately after fertilization. Figure 3 shows the results of one such experiment. It can be seen that considerable photoreactivation, as meas- ured by tso of the irradiated eggs approach- ing the control value, is effected in the first few minutes of illumination, but that after 25-30 minutes the cleavage delay is not further shortened by continuing illumination. (Indeed, there is some indication in this and other experiments that the cleavage delay may again be prolonged one or two minutes by the continuous BL.)

This type of experiment does not reveal whether the capacity for photoreactivation has been lost in the developing zygote 30 minutes after fertilization, or whether the UV lesion cannot in any case be photoreactivated to a greater extent than observed here. That both interpretations are to some extent correct can be seen from the second type of experiment with this system as summarized in table 4. Cleav- age in the control eggs at the temperature of this experiment ( 17°C) begins at about 75 minutes after fertilization. This 75 minute period was arbitrarily divided into three intervals of 25 minutes each, and the UV-irradiated eggs were illuminated during the first two (C) , the first only (D),

second only (E), third only ( F ) , or none ( G ) of these three periods. As before, illumination for 50 minutes (C) yielded no significant increase in photoreactivation relative to illumination for only 25 minutes (D). However, the results in (E) show that photoreactivation can still occur during the second 25 minute period, even though the extent of the recovery is some- what less. Hence the shortening of the cleavage delay seen in ( C ) and (D) corn- pared to zygotes kept in the dark, (G) represents maximum photoreactivation. 11- lumination during the third 25 minute period only ( F j produced only a small degree of photoreactivation. From experiments such as this, it would seem that the capacity for photoreactivation in the fertilization-first cleavage interval is limited, and that this capacity decreases with time after fertilization.

This limitation in the capacity of a UV- irradiated system to respond to “reactivating” illumination is sometimes expressed as the dose-reduction factor, DRF (Novick and Szilard, ’49 j. In other words, a maxi- mally reactivated population behaves like a population which has been given a smaller dose of UV and held in the dark. As indi- cated in the last line of table 3, eggs showing maximum photoreactivation after a UV dose of 8 X lo4 ergs/cm2 yield a curve for first cleavage which resembles that of eggs given 1/4 this dose and maintained in the dark. In this case, the DRF is therefore about 0.25. The constancy of this factor over a range of UV doses was not explored.

Included in table 4 are tso data for the second cleavage as well as the first, and the intervals between the tso)s for the first

TABLE 4

Time to 50% first and second cleavage of zygotes af ter UV-irradiation o f eggs and illumination of zygotes during various periods post-fertilization

UV dose = 8 X lo4 ergs/cm2. Temp. = 16°C. -~

Period of Dish no. uv fertilization I cleavage t50 I1 cleavage t50 AI-II post-

illumination

minutes none

._ 0-75

+ &25 + 25-50

+ none

._

+ 0-50

.+ 50-75

minutes 90 94 109 109 118 123 125

,minutes minutes 137 47 142 48 156 47 157 48 164 46 172 49 187 62

PHOTOREACTIVATION IN THE SAND DOLLAR 83

and second cleavage. It is to be noted that this interval is markedly prolonged (over that for the controls) for irradiated zygotes kept in the dark throughout. However, all populations which had been illuminated during any of the three periods prior to first cleavage showed virtually complete photoreactivation of the first - to - second cleavage interval, even in the case where the illumination came too late to have a significant effect on the delay in first cleavage.

DISCUSSION AND CONCLUSIONS

The data presented in the first section of Results are qualitatively very similar to those found in Arhacia by Blum et al. (Blum, Loos and Robinson, ’50; Blum, Robinson and Loos, ’51). They differ from the observations of Wells and Giese (’50) only in that these authors found Strongylo- centrotus sperm to be photoreactivated to a small extent when illuminated with a source very similar to our “Blacklight,” whereas we found Echinarachnius sperm to be only deleteriously affected at these wavelengths. The short-wavelength output of this source, in the vicinity of 0.32 LI, is absorbed to some extent by proteins and is effective at very high doses in the hemolysis of human erythrocytes (Cook and Blum, ’59). It is quite possible that, between the reactivating vs. inactivating effects of BL, one or the other predomi- nates at different dose or intensity levels. We have not explored this possibility. It is to be noted that Iverson and Giese (’54) were unable to demonstrate photoreactivation in the sperm of the echiuroid worm, Urechis caupo.

Our data, as well as those of the workers cited previously, are at a variance with those of Marshak (’49) in that he was not able to demonstrate photoreactivation in the unfertilized egg.

Marshak observed that if UV-irradiated sperm were not used immediately to fertilize normal eggs, the fertilization-to-first cleavage interval became even more prolonged as he protracted the time between irradiation and fertilization up to 80 minutes. The nature of this deterioration of UV-irradiated sperm was not further investigated. We were unable, at the dose level studied, to demonstrate more rapid

thermal reactions either ameliorating or worsening the UV effect. On the contrary, our results adhere closely to those expected from the “reciprocity law,” i.e., the radiation effect is a function of the total dose but is independent of the dose-rate.

The experiment presented in table 4, which is typical of several similar experiments, shows that while the capacity for photoreactivation decreases with time after fertilization, there is also a maximum degree of reactivation which is less than complete recovery. This observation is substantiated by the less-than-total photoreactivation of the unfertilized eggs (table 1) even before the development of the zygote is initiated. Less-than-total photoreactivation is the general observation in all systems where the phenomenon has been studied (Jagger, ’58). Our data indicate that the UV-induced delay in the first-to-second cleavage interval might be fully repaired by illumination prior to first cleavage. The determinations are not sufficiently exact, however, to be certain of a one or two minute difference, which would be a significant fraction of the 15 minute delay in the dark. Further, there has been one duplication of genetic material in this experiment, and with progressive cell divi- sions recovery from the delayed time- schedule of cleavage appears to occur even in the dark (Blum, Loos and Robinson, ’50). That photoreactivation does not com- pletely restore the UV-irradiated egg to its pre-irradiated condition is not surpris- ing in view of recent biochemical studies on UV-irradiated and photoreactivated DNA (Rupert, ’60; Marmur and Grossman, ’61). From such studies it appears that the radiation induces a variety of altera- tions in the DNA molecule, whereas the photoreactivating enzyme probably re- verses only one of these.

The events in the developing zygote which regulate the time-schedule of cleavage remain obscure. The careful studies of Fry (’36) on the rate of Arbacia development show that all cytologically observ- able stages are affected to the same extent by changes in temperature. Blum and Price (’50) found a definite ‘‘refractory period between fertilization and first cleavage, during which time UV-irradiation did not result in delay of first cleavage

84 JOHN S. COOK AND ALVIN F. RIECK

but did prolong the first-to-second cleavage interval. Comparison of these data with those of Fry indicates that the onset of the refractory period approximately coin- cides with the onset of prophase (see also Blum et al., ’54). Our data on the capacity of irradiated eggs to be photoreactivated (table 4 ) complement these observations. Fifty per cent cleavage of irradiated eggs, kept in the dark, was not reached for 125 minutes; nevertheless, 50 minutes after fertilization the delay in first cleavage was established and could be photoreactivated only to a very slight extent, while the delay in second cleavage responded to this reactivating illumination.

Recently Rustad (’59 b) found that acridine orange will delay first cleavage in Arbacia eggs during the first 20-25 minutes after insemination; later application of the dye had no effect on first but delayed second cleavage. In comparing his results to those of Blum and Price, he suggests (Rustad, ’59 a, b, c ) that the delay is an effect on the centriole or on a “nuclear trigger” for centriole replica- tion. We are now collecting cytological material to investigate such a possibility.

SUMMARY

1. Cleavage in the zygote of E. parma is delayed by UV-irradiation of either the egg or sperm. Illumination in the spectral range 0.3-0.5 will mitigate cleavage delay if applied to fertilized or unfertilized eggs which have been irradiated, or if applied to eggs which have been fertilized with irradiated sperm. Illumination of irradiated sperm prior to fertilization exacer- bates the ultraviolet effect.

2. Cleavage delay after sperm irradiation is independent of the dose-rate; no evidence was found for thermal reactions associated with the production of the UV lesion.

3. Maximal photoreactivation of the cleavage delay in irradiated eggs shows a dose-reduction factor of about 0.25.

4. Photoreactivation in irradiated eggs shows a “refractory period” during which illumination can no longer mitigate the delay in first cleavage; illumination during this period is effective in mitigating the prolonged first-to-second cleavage interval.

LITERATURE CITED

Blum, H. F., E. F. Kauzmann and G. B. Chapman 1954 Ultraviolet light and the mitotic cycle in the sea urchin’s egg. J. Gen. Physiol., 37:

Blum, H. F., J. S. Kirby-Smith and H. G. Grady 1941 Quantitative induction of tumors in mice with ultraviolet radiation. J. Natl. Cancer Inst., 2: 259.

1950 The accelerating action of illumination in recovery of Arbacia eggs from exposure to ultraviolet radiation. J. Gen. Physiol., 34: 167-181.

Blum, H. F., and J. P. Price 1950 Delay of cleavage of the Arbacia egg by ultraviolet radiation. Ibid., 33: 285-303.

1951 The loci of action of ultraviolet and x-radiation and of ahotorecoverv in the e m and sDerm

325-334.

Blum, H. F., G. M. Loos and J. C. Robinson

Blum, H. F., J. C. Robinson and G. M. Loos

of the sea -urchin Arbacia puniilata. 35: 323-342.

ibid.,

Cook, J. S., and H. F. Blum 1959 Dose rela- tionships and oxygen dependence in ultraviolet and photodynamic hemolysis. J. CeI1. Comp. Physiol., 53: 41-60.

Fry, H. J. 1936 Studies of the mitotic figure V. The time schedule of mitotic changes in developing Arbacia eggs. Biol. Bull., 70: 89-99.

Giese, A. C., D. C. Shepard, D. C. Bennett, A. Farmanfarmaian and C. L. Brandt 1956 Evi- dence for thermal reactions following exposure of Didinium to intermittent ultraviolet radia- tions. J. Gen. Physiol., 40: 311-325.

Iverson, R. M., and A. C. Giese 1954 Tests for photoreactivation in gametes of Urechis caupo. Science, 120: 504.

Jagger, J. 1958 Photoreactivation. Bact. Re- views, 23: 99-142.

Marmur, J., and L. Grossman 1961 Ultraviolet light induced linking of deoxyribonucleic acid strands and its reversal by photoreactivating enzyme. Proc. Nat. Acad. Sci., 47: 778-787.

Marshak, A. 1949 Recovery from ultra-violet light-induced delav in cleavage of Arbacia eggs by irradiation with visibie light. Biol. Bull., 97: 315-322.

Novick, A., and L. Szilard 1949 Experiments on light-reactivation of ultraviolet inactivated bacteria. Proc. Nat. Acad. Sci., 35: 591-600.

Rupert, Claude S. 1960 Photoreactivation of transforming DNA by a n enzyme from baker’s yeast. J. Gen. Physiol., 43: 573-595.

Rustad, R. C. 1959a Further observations re- lating radiation-induced mitotic delay to centriole damage. Biol. Bull., 117: 437.

The inhibition of mitosis in the sea urchin egg by acridine orange. Ibid., 117: 437438 .

1959c Induction of multipolar spindles by single x-irradiated sperm. Experientia, 15:

Wells, P. H., and A. C. Giese 1950 Photoreacti- vation of ultraviolet light injury in gametes of the sea urchin Strongylocentrotus purpur- a tus . Biol. Bull., 99: 163-172.

1959b

323-324.

studies on photoreactivation in gametes and zygotes of the sand dollar, echinarachnius parma

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