Kinetic Complexity, Homogeneity, and Copy Numberof … · 1470 ERSLAND ETAL. 404/435, secondary 470, dichroic reflector 460, and accessory secondary 530 (red attenuation filter)

Plant Physiol. (1981) 68, 1468-14730032-0889/81/68/1468/06/$00.50/0

Kinetic Complexity, Homogeneity, and Copy Number ofChloroplast DNA from the Marine Alga Olisthodiscus luteus1

Received for publication April 7, 1981 and in revised form July 14, 1981

DUNCAN R. ERSLAND , JANE ALDRICH3, AND ROSE ANN CATTOLICODepartment of Botany, University of Washington, Seattle, Washington 98195

ABSTRACT

The kinetic complexity of chloroplast DNA isolated from the chromo-phytic alga Olistwdiscw Iutes has been determined. Using optical reas-sociaton ques, it was shown that the plastid DNA of this algareacted as a single co eat with a second order rate constant of 4.1molar 1aIdsecond' I (Cot12 0.24molar second)undercondtions equivalentto 180 miliar Na' and 60°C. Given the 92 x 10* dalton complexitycalculated for this cbhooplast genome, an O sthodis cell contains 650plastome copies. Alhough this complement remains constant throughoutthe growth cycle of the organism, the ploldy level of an individual chloro-plast shows si nt plastity and is dependent upon the number ofchloroplasts present per cell. Experments with the DNA fluorochromeHoechst dye 33258 (bisbenzimde) demonstrate that plastids Isolated fromall phases of cell growth each possess a ring-shaped nucleoid containingdetectable DNA. Olihdwcus chloroplast DNA showed no sequencemismatch when thermal denaturation profiles of reassociated chloroplastDNA were examined, thus all plastome copies are essentially identical.Finally, reassociation studies demonstrated that no foldback (short invertedrepeat) sequences were present in the plastid genome altbough significanthairpin loop structures were observed in control nuclear DNA samples.

Reassociation kinetic analysis, contour length measurement,and restriction analysis demonstrate (2) that evolutionarily diverseplant types contain chloroplast genomes of approximately 90 x106 daltons in size. The plastids of all organisms studied to datedisplay a multicopy (polyploid) ctDNA4 content. Two studiesgave preliminary indication that the amount ofctDNA per organ-elle could vary within a multiplastidic cell. Panacium maximumwas shown (29) to contain 50 ctDNA molecules in bundle sheathchloroplasts but only 20 in mesophyll plastids, and it was reported(19) that Narcissus pseudonarcissus leaf chloroplasts contain 50ctDNA copies, whereas flower chromoplasts contain only 8ctDNA copies. The fact that ctDNA synthesis and chloroplast

' This work was supported by National Science Foundation GrantPCM7624440 to R. A. C. and United States Predoctoral Public HealthTraining Grant HDO7183 from National Institute of Child Health andDevelopment to D. R. E. and J. A.

2 Present address: Department of Horticulture, University ofWisconsin,Madison, WI 53706.

3 Present address: Standard Oil Company of Ohio, Research and De-velopment Laboratory, Cleveland, OH 44115.

4 Abbreviations: ctDNA, chloroplast DNA; CTAB, cetyl trimethylam-monium bromide; HAP, hydroxylapatite; nt, nucleotide; Cot, product ofmolar concentration of DNA nucleotides and time of incubation (M sec);CotU/2, the half reaction point in a second order DNA reassociation; Tm,melting temperature; ntp, nucleotide pair.

division are two separable events (see ref. 6 for review) which areresponsive to developmental and/or growth cues lent support tothe hypothesis of quantitative ctDNA plasticity. The unicellularalga, Olisthodiscus luteus provided an excellent system in whichDNA ploidy variation response could be analyzed within a non-differentiating cell. Using this naturally wall-less (8) test system,wherein precise chloroplast counts could be made, it was demon-strated (7) that the amount of ctDNA present per chloroplast wasinversely related to the chloroplast complement of the cell. Thisresult conclusively demonstrated that the chloroplasts of an or-ganism could be significantly plastic with respect to DNA com-plement. The observation made in the Olisthodiscus system hasnow been confirmed in higher plants. Expanding spinach (27) andmaturing pea leaves (18) were each shown to exhibit a similarinverse relationship between organelle DNA level and organellenumber. Moreover, since the genome sizes of the pea and spinachctDNA were known, it was possible to calculate the number ofDNA molecules present per plastid. Although the elegant studieson higher plant systems represented a major advance in ourunderstanding of the changes in organelle DNA ploidy levelswhich occur during different phases of plant growth and differ-entiation, difficulties of cell morphology (for example, the pres-ence of an intractable cell wall) necessitated protoplast isolation(18) or the use of Chl content (27) in ascertaining organellenumber. In addition, complexity of these higher plant systemsdictates that a measurement of organelle number reflects a changewithin a population of differing cell types. Thus, large variationsin organelle counts (18) per tissue sample are often obtained.A multiplastidic unicellular alga would represent an ideal sys-

tem to monitor DNA ploidy changes which occur when theorganism is subject to varying conditions of growth and/or devel-opment. The Euglena system which has been extensively analyzedshows (24) a dramatic change in the number of total ctDNAmolecules per cell when cultures are maintained under hetero-trophic, autotrophic, or mixotrophic growth conditions. Studies ofLyman and co-workers (personal communication) have shownthat the spectral quality of light significantly influences the levelsof ctDNA within Euglena. Cultures maintained under blue lightshow higher levels of ctDNA per cell than those cultures main-tained under a red light regime. Unfortunately, no information onplastid complement which is known to shift (22) in response tothe physiological state ofthe Euglena cell were done in conjunctionwith either of these studies. The macro-unicellular alga, Acetabu-lana, gives an exciting clue to the extremes ofctDNA complementpotential within the plastid. By use of the fluorescent dye DAPI,Coleman (11) has demonstrated that a large number of chloro-plasts have no DNA (or perhaps a DNA complement below thedetection level of this highly sensitive cytological technique). It isinteresting to note that the proportion ofAcetabularia chloroplastswhich contain detectable DNA is dependent upon the develop-mental phase of the organism.

In this report, ctDNA of the marine chromophyte 0. luteus is1468

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OLISTHODISCUS CHLOROPLAST DNA COMPLEXITY

characterized with respect to size and both intermolecular andintramolecular homogeneity. This simple unicellular alga may bemaintained axenically on a defined artificial sea water medium.Synchronization ofthe culture is induced by a 12 h light:12 h darkcycle, and the cells readily shift plastid complement in response toenvironmental cues. Knowing the size of the Olisthodiscus chlo-roplast genome allows monitoring ofctDNA molecules per plastidwhen this test system is subject to varying growth conditions. Todate, virtually no information is available on the control mecha-nisms involved in organelle biogenesis within chromophytic (Chla, c) plant systems.

MATERLALS AND METHODS

Culture Maintenance and Labeling. 0. luteus (Carter) was cul-tured in a defined artificial sea water medium on a 12 h light:12h dark cycle. Details of synchronous cell maintenance (8) and invivo [3H]adenosine labeling (12) have been presented elsewhere.DNA Extraction. Total Olisthodiscus DNA was separated (1)

into nuclear main band and satellite components by preparativelycentrifuging N-lauryl sarcosine cellular lysates in CsCl-Hoeschtdye 33258 density gradients. Main band nuclear DNA was useddirecly, whereas ctDNA required further cycles of CsCl-Hoeschtdye centrifugation to effect separation of this organelle DNA fromnuclear and second satellite (1) species. RNA and polysaccharidecontamination were removed from nuclear DNA by digesting thenucleic acid sample with RNase followed by CTAB precipitation(12). The DNA sample, dissolved in a 140 mm Na-phosphate (pH6.8) buffer, was then loaded onto a HAP column which wasmaintained at 60°C. The sample was eluted using 500 mm Na-phosphate (pH 6.8), then diluted to a final buffer concentration of120 mm Na-phosphate (pH 6.8) and centrifuged briefly to removeparticles of HAP. Chloroplast DNA was purified as describedabove, with omission of the CTAB step. Chloroplast DNA identitywas verified by fingerprint analysis using the restriction endonu-clease Eco R I (Aldrich, Gelvin, and Cattolico, manuscript inreview). The specific activity of 14 day in vivo labeled ctDNA was5,000 cpm/,ug. Labeled main band nuclear DNA had a specificactivity of 5,000 cpm/,ug.

Escherichia coli DNA was purchased from Sigma. This DNAwas dissolved in 0.1 M Tris (pH 9.0) and further purified by gentleshaking with an equal volume of 0.1 M Tris (pH 9.0)-saturatedphenoL followed by ethanol precipitation of the aqueous layer.Bacteriophage T4D (wild type) was the gift of A. J. Doermann(University of Washington). This DNA was purified by the hotphenol extraction technique of Massey and Zimm (20). Bacterio-phage lambda DNA was purchased from Bethesda Researchlaboratories and Bacillus subtilis DNA was a gift of R. Anderson(University of Washington). No further purifications of theseDNAs were necessary.Each DNA preparation was assayed (12) for purity by mea-

surement of thermal melt hyperchromicity and UV spectral qual-ity. Isolated Olisthodiscus DNA contains no impurities which arecapable (12) of significantly altering DNA reassociation rates. Tostore DNA, a preparation was dialyzed against 1 mm Tris, and thesample was then maintained at -20°C.

Shearing DNA. DNA samples contained in 200 mm NaCL 10mM Tris (pH 8.0), and 10 mm EDTA were sonicated (12) to aweight average length of 440 nt. Sonication was followed byextensive dialysis against reassociation buffer. Taking advantageof the observation (5) that DNA reassociation rate is affected bymonovalent cation concentration, different reassociation buffersystems were used, dependent upon the experiment, to precludethe use of large amounts of organelle DNA. The three buffersystems used in this study were: SSC (150 mm NaCl, 15 mmtrisodium citrate [pH 7.0]), 120 NaPB (120 mm Na-phosphate [pH6.8]), and 290 NaPB (290 mm Na-phosphate [pH 6.8]).DNA fragments larger than 440 nt in length were obtained by

mixing labeled nuclear or chloroplast Olisthodiscus DNA withunlabeled calf thymus DNA (Sigma) at a weight ratio of 1:500.This mixture was dialyzed against 140 NaPB buffer (140 mm Na-phosphate [pH 6.8]) and sheared to the desired fragment lengthsusing the methods presented in Table I. The DNA was then useddirectly for foldback reassociation analysis.

Thenmal Denaturatlon of DNA. DNA was denatured using aGilford 2400 recording spectrophotometer equipped with ther-moprogrammer and reference compensator attachments. DNAwas melted by raising the temperature at a rate of 1°C per min.Absorbance and temperature were recorded at 2-s intervals. Thethermal denaturation data were converted to probability plots asdescribed by Knittel et aL (17). The Tm of a sample is thetemperature at which 50% of the final melt hyperchromicity isobtained.

Reassociatlon of DNA. The reassociation of thermally dena-tured ctDNA was optically monitored using a Model 2400 Gilfordrecording spectrophotometer as described above. Observed reas-sociation rates were corrected for buffer sodium ion concentrationeffects (5) using a value of 1.0 for SSC and 120 NaPB or 3.62 for290 NaPB. Control experiments in which equal DNA concentra-tions were reassociated in different buffer systems resulted inidentical CotI/2 values after appropriate corrections were made.The data were plotted in Cot curve form (5) and fitted to optical(4) second order reassociation kinetics. All standard DNAs werereassociated using the 120 NaPB buffer system.To determine the proportion of foldback DNA sequences pres-

ent in either nuclear or ctDNA, the following procedure was used.Labeled Olisthodiscus nuclear or ctDNA mixed with calf thymusDNA (see above), sheared to varying fragment length, was dena-tured and cooled immediately to 0°C. These samples were thenpassed over individual HAP microcolumns, the bound DNAeluted, and the eluent counted using a Searle Isocap 300 liquidscintillation counter. Details of this HAP microcolumn techniqueare presented (12) elsewhere.

Cytoogical Visualization of ctDNA. Cultures were sampled inthe logarithmic or the linear growth phase at L6 of the synchro-nous cell cycle when neither cell nor chloroplast division is takingplace (8). When necessary, the sample was centrifuged at 1,500rpm for 2 min using a clinical table top centrifuge to concentratethe cells to approximately 106 cells/ml. A 4.0 #1 drop of thisconcentrate was placed on a washed glass slide and 1.0 tl Hoeschtdye 33258 (bisbenzimide) diluted to a concentration of 100 Wg/ml(stock solution 10 mg/ml H20) in Olisthodiscus 0-3 medium (7)was added. A coverslip was placed over the droplet and the samplewas allowed to dessicate slightly at room temperature. This treat-ment flattens the cells so that all chloroplasts can be observed (7)in one plane of focus. These cells were observed at a magnificationof l,OOOx, using a Zeiss 14 microscope equippped with a IVepifluorescence illumination and the following filters: primary

Table I. Methods Used to Shear Nuclear and Chloroplast DNA ofOlisthodiscus

Single'

Method Shearing Conditions Lcngth(nt)

Sonication 5PC, 5 Minb 440French press 20°C, 12,000 p.s.j.c 1,200VirTis 45 0°C, 30 Minc,d 3,000Hypodermic needle 20°C, 26-gauge needle, 10 passes 7,000' Weight average determined by alkaline agarose gel electrophoresis

(12).b Sheared in 100 mim EDTA, 200 mm NaCl, 10 mM Tris buffer (pH 8.0).c Sheared in 140 NaPB.d Conditions described in (12).

Plant Physiol. Vol. 68, 1981 1469



1470 ERSLAND ET AL.

404/435, secondary 470, dichroic reflector 460, and accessorysecondary 530 (red attenuation filter). Photomicrographs werethen taken using Kodak Tri-X film.

RESULTS

Reassociadon Analysis of ctDNA. Olisthodiscus ctDNA reas-sociates as a single second order component as demonstrated inthe typical experiment presented in Figure 1. The mean of sixreplicate experiments resulted in a Cot1/2 for Olisthodiscus ctDNAof 0.243 ± .026 M s. This DNA was reassociated at concentrationsranging from 10 to 20 ,ug/ml. Reassociation reactions were run atTm-250C in the appropriate buffer system, and the obtainedCot1/2 values were corrected (5) to equate with the Cot1/2 valueswhich resulted in a 120 NaPB buffer system. The reaction went toapproximately 80% completion because of the low concentrationofDNA (11 pg/ml) used in this experiment. At higher (20 ftg/ml)DNA concentrations in 290 NaPB, the reaction reached approxi-mately 85% completion (data not shown).To determine the size of the Olisthodiscus genome, this Cot1/2

value was compared to those obtained from a series of DNAstandards. Bacteriophage lambda, bacteriophage T4D, B. subtilis,and E. coli DNAs were reassociated in 120 NaPB buffer at theirrespective Tm.25°C criteria. A representative standard reassocia-tion curve (T4D) is presented in Figure 1. The molecular weights(daltons) used for these standards were 30.8 x 106 for bacterio-phage lambda, 185 x 106 for fully glycosylated wild type T4D,2,000 x 106 for B. subtilis, and 2,800 x 106 for E. coli, and themean Cot1/2 values resulting from our analysis were 0.088, 0.36,6.10, and 8.60 M s, respectively. When the Cot1/2 for OlisthodiscusctDNA is plotted (Fig. 2) against the Cot1/2 values obtained forthese standard DNAs, a value of 92 x 106 d is obtained. Thisvalue is in agreement with determinations of Olisthodiscus ctDNAcomplexity obtained by contour length measurements (1) andrestriction endonuclease digestions (Aldrich, Gelvin, and Catto-lico, manuscript in review) which were 95 x 106 d and 99 x 100 d,respectively.ctDNA Molecules per Organelle. An Olisthodiscus cell (7) con-

tains a constant level (0.103 x 10-12 g) of ctDNA, even thoughchloroplast number shifts in response to growth parameters. If amol wt of 95 x 106 d is used (mean of the contour length,restriction, and reassociation analysis) to calculate the number ofchloroplast genomes, then an Olisthodiscus cell maintained underphotoautorophic conditions will contain approximately 650ctDNA molecules both in the logarithmic and in the stationary

k,

0

50

1004 -3 -2 -l 0

log CoTFIG. 1. Reassociation of Olisthodiscus chloroplast DNA. (0), chloro-

plast DNA was reassociated (11 ,ug/ml) in 290 mM NaPB at 57.50C. The

corrected Cot1,2 (0.061 M uncorrected value x 3.62) was 0.235 M for thesingle second order reassociation component. (A), control T4D DNA (10,tg/ml) reassociated under the same conditions described for chloroplastDNA. The uncorrected Cot1/2 was 0.0985 M s.

cJ

._3

crJ0)0

-i

Plant Physiol. Vol. 68, 1981

10

Log Molecular Weight (daltons)

FIG. 2. Determination of Olisthodiwsu chloroplast DNA kinetic com-plexity. (0), standard DNA preparations (440 nt weight average length).Reassociated at respective Tm.250C in 120 NaPB. Each equivalent Cot1/2point shown represents an average of three determinations. DNA concen-trations were 4 to 10 pg/ml (A and T4D) and 8 to 100 Ag/ml (B. subtilis,E. coli). (0) chloroplast DNA (440 nt weight average length). This datapoint represents an average of 6 Cot,/2 determinations. Identical Cot1/2values were obtained after correction ("Materials and Methods") for cationconcentration in SSC, 120 NaPB, or 290 NaPB buffer systems. Eachchloroplast DNA reassociation was conducted in parallel with a standardDNA (A, T4D, or B. subtilis) to control for deviations in reassociationconditions which occur between experiments.

C,)j40 o0~~~~0 0

01 30C.)w 0 00

0-o 20z°0\

O o

12 16 20 24 28 32 36 40CHLOROPLAST NUMBER

FIG. 3. Inverse relationship between ctDNA molecules and chloroplastnumber per cell. Chloroplast number and absolute ctDNA content per cellwere obtained from Cattolico (7).

phases of growth. Therefore, an Olisthodiscus chloroplast is plasticin DNA complement (Fig. 3), having as high as 44 and as low as13 ctDNA molecules per organelle, dependent upon culture age.As seen in Figure 4, bright circlets of ctDNA can be seen when

9

0. kitaus , T4 DNAcNoroplast DN ik

I




B

FIG. 4. Fluorescence visualization of Hoescht dye 33258-stained Olis-thodiscus. Light micrograph oftwo cells obtained from a logarithmic phaseculture. Cells in this culture contained a mean chloroplast number of 31.N, nucleus; CN, chloroplast nucleoid. Bar is 5 t&m. A, cell from suspensionfreshly placed on glass slide. Chloroplast nucleoid can be seen on edge(arrow). B, cell flattened into single plane of focus.

Olisthodiscus cells are stained with Hoescht dye 33258. Approxi-mately 1,000 chloroplasts from logarithmic and from stationaryphases of growth were analyzed for DNA content. A scorablefluorescent response was obtained in 99.6% of all organellesexamined. This observation indicates that every chloroplast withinan Olisthodiscus cell contains DNA.

Intermolecular Homogeneity. Given the large number ofctDNA molecules per organelle, the probability of heterogeneity(sequence divergence) due to random mutational events couldeasily exist. To determine whether the Olisthodiscus ctDNA mol-ecules were homogeneous or heterogeneous, a comparison wasmade between the thermal denaturation profile of sheared nativectDNA (control) and sheared ctDNA which had been melted thenreassociated in SSC buffer to Cot 0.98. For every 1% mismatch insequence resulting from imperfect strand pairing during formationof duplexes, a 1°C depression in Tm would occur (5). As seen inFigure 5, both native ctDNA and sheared ctDNA show an iden-tical profile which demonstrates that no extensive heterogeneity ispresent among the multiple ctDNA molecules which exist withinan Olisthodiscus cell. The calculated ATm (difference in Tm be-tween native and reassociated DNA) for Olisthodiscus ctDNA,representing the mean of three experiments as -0.2°C 0.12.This value is slightly higher than the ATm seen (Fig. 5) for phageA DNA (-0.50C) indicating that, given the sensitivity of thistechnique, limited microheterogeneity could exist within the Olis-thodiscus ctDNA population.

Intramolecular Homogeneity. To determine whether Olisthod-iscus ctDNA contained inverted repeat sequences (foldback se-quences) which would reanneal to form intrastrand duplexes (5,

90 7

:~80-E 70:*60:50

>.-40

-0o 20-

10

70 76 78 80 82 4 8 9 2 9Temperature (°C)

FIG. 5. Thermal denaturation ofnative and reassociated DNA samples.Chloroplast DNA: (-), melt ofnative DNA, (0), melt ofCot 0.977-duplex.Bacteriophage A DNA: (A), melt of native DNA, (A), melt of Cot 1.345-duplex.

30-

) 20

I10

0

2 3 4 5 6 7FRAGMENT LENGTH (ntp X 10-3)

FIG. 6. Zero time binding of 3H-labeled Olisthodiscus nuclear andchloroplast DNA. Double-stranded DNA regions were assayed using Cot6.0 x 10' DNA samples which were of increasing fragment length (TableI). All points are averages of duplicate determinations. (A), nuclear DNA;(0), chloroplast DNA.

13), in vivo labeled ctDNA was sheared, denatured by boiling, andthen immediately cooled to 0°C. The low (6 x 10-6) Cot attainedby this method is sufficient to allow only self-complementarysequences to reassociate. The DNA is then subject to HAP chro-matography and the proportion of double-stranded DNA presentwithin each sample assayed. If foldback sequences do exist andare separated from one another along the DNA molecule, then itis expected that as DNA fragment size increases, the proportionof total DNA which binds to HAP will also increase. The presenceof one duplex of minimal length (only 45 ntp are required forHAP binding to occur) would thus be sufficient to insure bindingof an entire labeled DNA fragment. The data obtained in Figure6 demonstrates that little foldback occurs in the Olisthodiscuschloroplast genome when DNA fragments of 440 nt to 7,000 nt inlength were analyzed. Within the longest fragment size assayed(7,000 nt), zero time binding (HAP binding of DNA at Cot 6.0x 10-6) only 4% of the total DNA counts applied to the HAPcolumn, and at 440 nt fragment size, the zero time binding fraction(2.3%) is almost identical to the level of nonspecific single stranded

--

S

Plant Physiol. Vol. 68, 1981 1471




DNA HAP binding (1.5%) which normally occurs in this assay.For example, in a control study, 98.5% of the radioactivity ofdenatured "C-labeled 400 nt length E. coli DNA does not bind toHAP at 60°C in 140 NaPb. In addition, the behavior of anOlisthodiscus nuclear DNA control in these experiments showeda significantly different result than that obtained with the ctDNAspecies. The zero time binding of labeled nuclear DNA (Fig. 6)increased as a function of fragment length such that 24% of the7,000 nt fragments contained at least one foldback sequence.

DISCUSSION

Reassociation kinetic analysis demonstrates that chloroplastDNA present within the alga 0. luteus is 92 x 106 d in size. Thisvalue agrees well with ctDNA size determinations obtained (1;Aldrich, Gelvin, and Cattolico, manuscript in review) for thissame organism using contour-length measurements (95 x 106 d)of relaxed supercoiled molecules and restriction enzyme digests(99 x 106 d) of Hoescht dye separated satellite DNA obtainedfrom isolated chloroplast preparations. This information repre-sents the only published experimental data for ctDNA size amongthe chromophytic plant (Chl a and c) evolutionary sequence. It isinteresting to note that the ctDNA size of Olisthodiscus is similar(2) to those reported among several chlorophytic (Chl a and b)phyla analyzed. This observation suggests that conservation ofctDNA size occurs among a variety of plant types, regardless ofevolutionary origin. The reason for this relatively stringent sizemaintenance is presently unknown.An Olisthodiscus cell contains approximately 650 ctDNA mol-

ecules. To date, among the numerous multiplastidic cells, totalctDNA copy number has been reliably determined (9, 18, 21, 27)only for Euglena, spinach, pea, and mung bean. In all cases, thectDNA copy number is high (between approximately 350 and5,000 chloroplast genomes per haploid nucleus). In Olisthodiscus,a difference in chloroplast and cell division rate combined with atight maintenance of nuclear:chloroplast DNA ratio (7), results ina shift in plastid genome copy number as the organism progressesthrough various phases of the growth cycle. As a result, a logarith-mic phase cell contains a high chloroplast complement but fewctDNA copies per chloroplast, whereas cells in stationary phasedisplay a decreased organelle level and a high ctDNA content perchloroplast. Although the nature of the methods employed in thisstudy allow us to discuss this finding only in terms of averagectDNA copy numbers, these shifts represent a real phenomenon.The changes observed can not be explained by postulating that alarge fraction of the chloroplasts are devoid ofDNA, for as shownby our Hoescht dye studies, virtually every plastid contains adetectable ring-shaped nucleoid. In systems such as Acetabularia,wherein no ctDNA is observed (11) in many plastids, and inhigher plants which display an array of plastid types (chromo-plasts, amyloplasts, chloroplasts, etc.) within a single organism, itis obvious that the definition of plastid functionality and/orviability may vary significantly. The events of ctDNA replication,ctDNA partitioning, and plastid division must be mechanisticallyrelated. How these events relate to chloroplast genome expressionand thus chloroplast maintenance during cell growth and differ-entiation remains an intriguing question.

Melting point depression analysis indicates that no significantheterogeneity in sequence composition exists in the multicopyOlisthodiscus ctDNA population. In this report, control experi-ments using bacteriophage DNA which is highly homogeneous insequence composition have demonstrated that 0.5% sequencemismatch in ctDNA or a heterogeneity in 4.75 x 105 d (720 ntp)would not be detected by this thermal denaturation technique.Microheterogeneity has been observed (Aldrich, Gelvin, and Cat-tolico, manuscript in review) in restriction endonuclease digests ofOlisthodiscus ctDNA. This microheterogeneity in the ctDNA pop-ulation is stable from experiment to experiment and through all

phases of the growth cycle.Hydroxyapatite binding of 3H-labeled Olisthodiscus ctDNA at

Cot 6.0 x 106 revealed little ifany foldback sequence. In contrast,the zero time binding fraction in the nuclear DNA control in-creased as a function of fragment length. To date, few studies offoldback sequences within plastid DNAs have been reported. Ithas been observed that leucoplast DNA of the colorless chloro-phyte Polytoma obtusum contains a substantial foldback fraction(28) and recent nucleotide sequence data demonstrate that aninverted repeat exists in the middle of a 16S ribosomal RNA genefrom corn ctDNA (26). This inverted repeat would be of sufficientlength to be scored as a foldback sequence in a HAP bindingassay. Finally, small inverted repeat sequences 100 to 300 ntp longhave been observed to occur at defined positions in the chloroplastgenome of Chlamydomonas reinhardtii (14, 25). Gelvin and Howell(14) have speculated that these inverted repeats may function asregulatory elements which lie adjacent to coding sequences. Theregulation of gene function in several bacterial systems is effectedby inverted repeat (palindromic) sequences which are muchshorter than the presumptive control sequences observed inChlamydomonas ctDNA. For example, short (10-15 ntp) palin-dromes act in transcription initiation (15) and in the attenuationmechanism in operons responsible for amino acid synthesis (16).Such repeats might also exist in Olisthodiscus ctDNA but are notrecovered by our HAP assay technique. A minimum length ofapproximately 45 base pairs is necessary for binding of double-stranded DNA to HAP under the conditions chosen for our study(600 in 140 NaPB). If foldback DNA is shown to have a regulatoryrole in ctDNA expression, then we must explain why some plastidgenomes contain foldback sequences in higher proportion and ofgreater length than those found in prokaryotic systems.

It should be noted that zero time binding results do not rule outthe presence of a large inverted repeat structure in OlisthodiscusctDNA. In corn ctDNA, a 22,000 ntp sequence containing thechloroplast ribosomal RNA cistrons is repeated in an invertedorientation and the repeats are separated by a nonhomologousDNA sequence 12,500 ntp long (3). If Olisthodiscus ctDNA wasorganized in this manner, labeled ctDNA fragments at least 12,600ntp in length would be necessary to detect a large inverted repeatwhich would appear in a zero time binding experiment as a shortdouble-stranded stem attached to a large single-stranded loop.

In conclusion, parallels in plastid behavior at the genetic (6)and physiological (23) levels between Olisthodiscus and other plantsystems demonstrate that this marine chromophyte can serve as amodel system for the elucidation of events which occur duringchloroplast growth and division.

LITERATURE CITED

1. ALDRICH J, RA CArroLIco 1981 Isolation and characterization of chloroplastDNA from the marine chromophyte Olisthodiscus luteus. electron microscopicvisualization of isomeric molecular forms. Plant Physiol: 68: 641-647

2. BEDBROOK JR, R KOLODNER 1979 The structure of chloroplast DNA. Annu RevPlant Physiol 30: 593-620

3. BEDBROOK JR, R KOLODNER, L BoGoRAD 1977 Zea mays chloroplast ribosomalRNA genes are part of a 22,000 base pair inverted repeat. Cell: 739-749

4. BENDICH AJ, RS ANDERSON 1977 Characterization of families of repeated DNAsequences from four vascular plants. Biochemistry 16: 4655-4663

5. BirsrEN RJ, DE GRAM, BR NEuPsLD 1974 Analysis of repeating DNAsequences by reassociation. Methods Enzymol 29E: 363-406

6. BUTrERFASST 1979 Patterns ofchloroplast reproduction. Springer-Verlag, Wien,New York, pp 71-74

7. CArrOLICo RA 1978 Variation in plastid number: effect on chloroplast andnuclear deoxyribonucleic acid complement in the unicellular alga Olisthodiscusluteus. Plant Physiol 62: 558-562

8. CATTOLIco RA, JC BOOTHROYD, SP GiBBs 1976 Synchronous growth and plastidreplication in the naturally wall-less alga Olisthodiscus luteus. Plant Physiol 57:497-503

9. CHELM BK, PJ HOBEN, RB HALLICK 1977 Cellular content of chloroplast DNAand chloroplast ribosomal RNA genes during chloroplast development. Bio-chemistry 16: 782-786

10. CHRISTIANSEN C, G CHRISTIANSEN, A LETHBAK 1973 The influence of glucosyl-ation on the renaturation rate of T4 phage DNA. Biochem Biophys Res

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Commun 52: 1426-143311. COLEMAN AW 1979 Use of the fluorochrome 4'6-diamidino-2-phenylindole in

genetic and developmental studies of chloroplast DNA. J Cell Biol 82: 299-305

12. EasxLAN DR, RA CATTOLIco 1981 Nuclear DNA characterization ofthe marinechromophyte Olisthodiscus luteus. Biochemistry: In press

13. FLAvELL R 1980 The molecular characterization and organization of plant-chromosomal DNA sequences. Annu Rev Plant Physiol 31: 569-596

14. GELvIN SB, SH HowmuL 1979 Small repeated sequences in the chloroplastgenome of Chlamydomonas reinadii Molec Gen Genet 173: 315-322

15. JovmN TM 1976 Recognition mechanisms of DNA-specific enzymes. Annu RevBiochem 45: 889-920

16. KEu.LE EB, JM CALVO 1979 Alternative secondary structures of leader RNAsand their regulation of the trp, phe, tris, thr, and lev operons. Proc Natl AcadSci USA 76: 6186-6190

17. KNTrEL MD, CH BLAcI, WE SANDINE, DK FRASER 1968 Use of normalprobability paper in deternining melting values of desoxyribonucleic acid.Can J Microbiol 14: 239-245

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Kinetic Complexity, Homogeneity, and Copy Numberof … · 1470 ERSLAND ETAL. 404/435, secondary 470, dichroic reflector 460, and accessory secondary 530 (red attenuation filter)