Download milliliters per liter. After 5-day-old cultures wvere

ASSIMIILATION OF ANIMONIA BY NITROGEN-STARV ED
CELLS OF CHLORELLA \"ULGARIS 1
JAMES E. BAKER2 & JOHN F. THOMPSON
U. S. PLANT, SOIL, & NUTRITION LABORATORY. AGRICULTURAL RESEARCH SERV[CE.
U. S. DEPART-MENT oF AGRTCULTURE, ITHACA, NF\V YORK
5 days at
IN1TRODUCTION
in an investigation of the utilizationi of inorganic
nitrogen by Chlorella, Reisner- et al (17) measured
the changes in the quantities of unlcomiibined amino
acids whiclh occurred after a(l(ling nitrate or ammlilonia
to nitrogen-starve(d cells. Ad(ling ammllolnia produced
more rapid and nmore striking changes in amounts of
amino aci(ds than ad(ling nitrate. InI the shortest period of ammnioniia treatment (15 mmiin) mlarkedl increases
in glutamiline a(dl alaninie aln(d a simall inlcr-ease in selrinle
were observed.
To leal-rn miiore about the slhort-term-l assimilation
of ammolnia by Chlorella, the followving investig,ations
wrere carrie(l out: A The colncelntration of several
uincomliiied amiino aci(ls in Chlolrella -was measured
2 to 15 minui1tes after adding anmmiioniumni chloride:
B : The effects of inhibitors on tlle concenitration of
amin;1o acids, aftel- a(lding amimionia, Nwere determined;
C: Enzymies that mliglht be involved in ammonia
utilization wTere stu(lied; D The amiiounlt of N15 in
noni-proteini alaninie, glutalnine, ancd glutamate was
miieasured after administering N15H,NO:3 for 1 and 5
mlinute intervals; E: The concentrationi of pyruvate
and a-ketoglutarate vas (letermined before and after
adding ammonia to Clhlorella: F: The effect of
pyruvate and ammonia plus pyruvate on the qtuantity
of aniino acids wvas exaamined.
a
packed-cell volume (16) of about teni
milliliters per liter. After 5-day-old cultures wvere
centrifuged at 2,000 g for 15 iminutes, the supernatanit
solutioin -was carefullyN remov-ed. The cells wer-e resuspen(led in the supernataint solution (whiclh lhad
been autoclaved & coole(l) to give a concelntrate(d Uiniform suspension containing approximately 1.5 ml of
cells per 10 mfl. Eachi salillple comipr-isedI 10 mil of
suspensioln.
EXPERIAIEN'I Al,
ella
PROCEDURES: Additions to Chlor-
suspenlsiolns w-ere miia(le in tw o ways. In soniie
experimnen1ts, anI initial control samiple w-as takeni and
the remiiainiing stuspen sion wN-as poure(d imnmediatelyv
into a flask conitainiing (lrv miaterial. Furtlher 10-nill
aliquots of suspensioln were r-emovedl at various timlles
after inixing. In otlher experimients, a concelntrate(l
solutioni of test coniipound was added to the suspelnsioni.
Ininmediately after niiixing, oine 10-niil portion vas
zero-tiimne samiiple an(i subsequent 10-il1
the samne way. During the
incubation period, cells wvere sllakein in light at 210 C.
Separate 0.1 5-nil ali(luots \w ere takeili to (leter-illie
packed-cell voluniie (16).
For amino acid (leteriiiinationis. 10-nil aliquots of
cell suspension were stirre(d into 50 nil of 95 % ethanol
at -78° C. After warmiing to rooiii temperature, the
mixture was ceiitrifugedl and the supernataiit solution
was poured off. The resi(lue was re-extracte(d witl
five successive 50O-nil batches of 80 % ethaIiol at rooiii
MATERIALS & METHODS
STOCK CULTURE & INOCULATION: Chlorella vitl- temperature. The combiniedl supernataiit solutions
evaporated to dryness in v-acuo oii a rotary evapgaris Beijerinck var. viridis (Chodat) was obtained were
at 300 C alnd the resulting resi(lue was dlisfrom the University of Indiania algal collection and orator
solved
in water. The extracte(h aiiiiiio acids were
maintained as described previously (17).
purified on ion exchange resiIis (20) and quantitativeGROWTH OF NITROGCEN-STARVED CHLORELLA & ly assayedl by paper chromiiatography (19).
In enzyme studlies, Chlorella cells were separated
PREPARATION OF CONCENTRATED CELL SUSPENSIONS:
Chlorella was grown on a low-nitrogen medium (17) froni the nutrient solutioin by centrifugationi. The
containing 1 % glucose. One liter of sterile nutrient residual cell paste was weiglhed and cooled. Aftersolution in a 4-liter Erlenmeyer flask was inoculated the cells were grouIid witlh five times their weight
aspectically and shaken in continuous light (50 ft-c) of levigated aluniina in a chilled mortar, the resultaiit
on a reciprocal shaker (30 cycles/min) at 210 C. mixture was extracted with three volumes of cold
Lack of nitrogen resulted in a cessation of growth in 0.2 i-a tris3 buffer at pH 8.15. The suspension was
as a
The following abbreviations are used in this paper:
tris(lhydroxyniethyl) aminomethane; ATP =
adeiiosiiie triphosphate; DNP = 2,4 dinitrophenol; DOP
= deoxypyridoxine; DPNH = reduced diphosphopyridine
tris =
' Received September 23, 1960.
2
remove(l
samllples wNere obtained in
Present address: Agricultural Marketing Service,
U. S. Department of Agriculture, Beltsville, Md.
nticleotide.
208
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BAKER &
THOMPSON-NH4
ASSIMILATION OF
centrifuged and the supernatant solution was dialyzed
over-night at 60 C against 0.001 M glutathione. The
protein content was estimated by the method of
Lowry et al (12).
Glutamic dehydrogenase was assayed by the Bulen
method (1). Tests for alanine dehydrogenase were
made by a similar method (22).
Glutamic acid-alanine and glutamine-alanine transaminases were measured by the incubation of dialyzed
extract with glutamic acid plus pyruvic acid or glutamline plus pyruvic acid at 370 C for 60 or 120
minutes. The glutamic acid or glutamine and alanine
were determined by reaction with ninhydrin on paper
(19) after separation by one-directional paper chromatography in phenol and water (8: 3 v/v). Glutamic acid-serine transaminase assays were made in
a similar manner using hydroxypyruvate prepared
from pyruvic acid (5).
Glutamine synthetase was measured by the method
of Elliot (6).
Tests for the formation of alanine from pyruvate
and adenyl amidate (2) were made by the procedures
of Katununia (9).
In experiments with N15, the uncombined amino
acids were extracted from cells with ethanol as described above. The amino acids from an entire
sample were purified and chromatographed on 12
chromatograms (19). The amino acids were measured on one chromatogram by the quantitative ninhydrin reaction (19). The other chromatograms
were sprayed with a 0.05 % ninhydrin solution in
methanol. After 10 minutes at room temperature the
glutamic acid, alanine, and glutamine spots and blank
areas of paper of corresponding size were cut out
(the other amino acids were omitted). The weight
of paper was determined in each case. The paper
pieces were dipped in KOH-borate buffer (4) and
aerated at 400 C for 10 minutes to remove ammonia.
The paper pieces were then eluted with 50 % ethanol.
The eluate of the glutamine spots was hydrolyzed
with 1 N HCl for 2 hours at 1000 C. The hydrolysate
was made alkaline and the ammonia from the amide
group was distilled into HCl. The remaining glutamic acid was digested with sulfuric acid as were
the eluates of glutamic acid and alanine spots. The
total ammonia in the digests and distillate was determined on a separate aliquot with ninhydrin (4).
From eluates of blank areas on the chromatograms
treated in the same manner, the contaminating nitrogen in the paper was determined. The remainder of
the digests were made alkaline and the ammonia was
distilled into 0.1 N HCl. The NH4Cl obtained from
the digestion of amino groups and from the hydrolysis
of amide groups was converted to nitrogen with
NaOBr and the N15 content was measured in a mass
spectrometer.
Keto acids were extracted and converted to their
dinitrophenyl hydrazones and the hydrazones purified
by the procedures of Isherwood and Niavis (8).
Appropriate portions were chromatographed on paper
using n-butyl alcohol and NH4OH (15). The two
N-STARVED
209
CELLS
pyruvic acid spots and a-ketoglutaric acid spots were
eluted with 1 % Na2CO3 solution (10).
RESULTS
It was found previously that addinig ammonium
chloride to nitrogen-deficient Chlorella (17) resulted
in a large increase in the content of non-protein alanine after 15 minutes. To learn if the alanine increment was apparent in shorter times, ammonium
chloride was given to Chlorella for 2, 5, 10, and 15
minute intervals. Four amino acids showed significant changes (table I). In the shortest time (2
min) the alanine content showed the greatest increase
of any acid both on an absolute and on a percentage
basis. The synthesis of alanine appeared to take
place immediately after adding ammonium chloride.
Within 15 minutes, glutamic acid decreased and
then returned to its initial level. The decrease was
certainly a consequence of glutamine synthesis. The
increase in glutamine more than offset the decrease
in glutamic acid resulting in a net increase in the
total of glutamic acid plus glutamine.
Since it might be expected that DNP would inhibit
glutamine formation and that DOP would inhibit
transamination (23), the effect of these inhibitors on
the assimilation of ammonia was examined (table I).
TABLE I
UNCOMBINED AMINO ACIDS IN CHLORELLA BEFORE &
AFTER ADDING AMMIONIUM CHLORIDE
AMINO
,umoles/ml
ACID
MIN
0
OF CELLS
AFTER AMMONIA
2
5
ADDITION*
10
15
Glutamic acid
1.22 1.24 1.14 1.17 1.27
Glutamine
0.23 0.40 0.75 1.73 2.04
Glutamic + glutamine 1.45 1.64 1.89 2.90 3.31
Alanine
0.69 1.20 1.83 2.66 2.98
Serine
0.51 0.55 0.56 0.69 0.77
After 30 w1in incubation with 10-4 M DNP
Glutamic acid
1.00 ... 0.92 ... 1.16
Glutamine
0.13 ... 0.84 ... 0.92
Alanine
0.79 ... 1.64 ... 1.96
120
wnin incubationt with 10-4 M DNP
After
Glutamic acid
0.97
0.82 ...
1.05
Glutamine
0.15
0.89 ... 0.98
Alanine
0.80
1.53 ... 1.92
After 30 min inscubation zeith 3 X 10-3 M DOP
Glutamic acid
1.40 ... 1.07
1.21
Glutamine
0.16 ... 1.50
2.42
Alanine
0.55
1.96
2.93
After 120 mimt incubation with 3 X 10-8 M DOP
Glutamic acid
1.29 ... 1.07 ... 1.14
Glutamine
0.15 ... 1.40 ... 2.04
Alanine
0.42 ... 2.09 ... 3.08
* 60 1umoles of NH4Cl given per ml of cells.
...
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210
PLANT PHYSIOLOGY
Altlhouglh DNP (lid lot l)revent the increase of alanine
an(l glutaamine between 0 and( 5 iminutes. changes in
these aci(ds between 5 and( 15 minutes were much
re(luce(l. Tn the presence of DOP, glutainiiie was
highier at 5 nminutes aln(d the same at 15 imlinutes as the
untreatedl cells. These results are (lifficult to interpret. DNP did niot inhibit glutainiiile forilmation in
5 iminlutes but did between 5 and 15 mllinutes irrespective of its time of conltact with cells. Although DOP
did niot inhibit alanine form-lationi this (loes nlot prove
that alaninie is not formed( b)v transaniination.
TABf,I. II
FoRNrFA-1IONx OF AL,AN-INF FRO\r GLUTA-MATE & PYRUVATE
IN PRESENCE OF CELL-FREE EXTRACT FROM CHLORELLA
REACTION MIXTURE
,umoles ALANINE FORMED/
Complete*
Minus pyruvic aci(d
0.37
0.0
0.0
nmg PROTFIN /IR
Pltis boiled extract
* The complete reaction mixture contained 6.0 ,smoles
Na-pyrtuvate, 6.7 ,umoles Na glutamate, 100 gnmoles tris,
anid ca. 1 mig protein from the extract in a total volume
of 2 mln. The mixtures were incubated for 1 lhouir at 370 C
on a Dtihntoff shaker.
Enzymnes whichi imiiglht be responsible for the
changes in amiino acid content following ammonia
addition, were assaye(l. Chlorella extracts have glutamline dehydrogenase (0.21 Atmoles DPNH oxidized
min/mig protein), glutaimiic-alaninie transhydrogenase
(table II) (cf. 15) anid( glutaminie synthetase. No
eyidence wvvas obtained for alanine delyd(lrogenase, glutamic-serine transamiiinase, glutamine-alanine transamiinase, and aniimiation of pyruvate with adenyl
amlidlate (9).
If alanine were form-ied by direct amiination of
pyruvate, adding labele(d amimionia (N' 53) to Chlorella
should result in a high N' conceintrationi in alanine.
N' 5HNO0, was given to nitrogen-starved Chlorella
cells for 1 an(l 5 minllte intervals. Table TII gives
TAlWE III
CONCENTRATION & N15 CONTENT OF
A'MINO ACIDs OF CHLORELLA
ADDING N"-H 4NO-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~:
AFTER ADDING
AMIINTO ACID MIN
1
5
0
p.oiioles of Anulitfo
acid/liil of cells
Glutamic acid
Alaninie
Glutaminie
0.84
0.53
0.09
0.63
0.89
0.32
0.48
2.18
0.59
UNCO-MBINED
AFTER
Ni 5H NO *
1
5
atomt % excess
of N15
15.8
6.5
33.8
20.7
Amino
group
16.2
44.4
36.8
59.8 _
Amide
group
60 atom
%,
of
excess
of N
H,NO1 was used.
TABLE IV
PYRUV IC ACID & a-KETOGLUTARIC ACID CONTENT OF
CHLORELLA BEFORF & AFTER ADDING
AMM:\ONIIUM\ CHII
/unioles/imil CELLS
TImE AFTEIR
ADDING
H4CI*
0
5 min
15 Im1in
> 60 umoles
ORIDE
PYRUN IC ACID
a-KETOGLUTARIC
0.35
0.16
0.17
0.23
0.51
0.51
per
ACID
ml of cells.
the atoimi per cent excess of N' in uncomlibined alaniine,
glutanuine, and( glutamiiic acid as wvell as the quantities
of these acids. The notahle point is that alanine has
a muclh lower N'i conitent than glutamic aci(l. Tllis
result suggests that alaninle is fornlle(l by transamination from glutamic aci(l.
If alanine is forme(d bv transamination its rapi(l
rise after addiing ammiloni<a coul(l result fromii aii increase in pyruvate or a dlecrease in a-ketoglutarate
(since glutamic acidl (loes not chlanige miiuch). The
keto aci(d content of Chlorella before anid after ad(ling
anmmiiolnia was determinedl (table IV). Pyruvic acid
level inicrease(d about 50 C% and( a-ketogluitaric acid to
a lesser extent.
TABLE N'
FFFECT OF PYRLvICcACID & AM]MON-NIA ON CONTF.NTI
OF UNCOMBINED ALAN-INF, & GI.UTAMIC
ACID IN CILOREI.xA
TIME IN MIIN AFTER
ADDING INDICATED COMPOUNDS CONTENT OF AMINO ACIDS
PYRUVIC ACID*
'NH,CI*
0
30
32
35
45
0
0
0
60 unmoles/nml
0
0
2
5
15
2
5
15
of cells.
GLUTAMIC ACID
ALANINI
,uimoles /m,,l of cells
0.86
0.73
0.88
0.81
1.24
0.67
1.94
0.59
2.23
1.26
0.93
0.73
1.39
0.64
2.98
0.97
Since pyruvic acid increase(d on the adldlitioni of
amimioniaC, the effect of adding pyruvic acid eitlher
without or with anmmionia on the alanine level was
(letermine(l. Data oIn alanine an(l glutamic acidl only
are included since glutamic acid is the most likely
(lonor in transaniination to alainine (table V). Althouglh the a(lditioni of pvruvic aci(d increased the alaiiiiie content and(I (lecrease(l the glultamiic acid content
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BAKER &
THOMPSON-NH4 ASSIMILATTON
somiiewhat in 30 minutes, it is clear that the prior
incubation with pyruvic acidl cloes not change the
extent of the subsequent increase in alanine content
after the addition of ammonia.
DiscuSSION
The central role of glutamiiic acid in nitrogen metabolism of plants has been emphasized repeatedlly
(3. 11, 24, 25). After tlle feeding of N'5-labeled
ammoniia to plants, the relatively high N'5 content of
glutaniic acid has been noted (13, 21, 25). More
recent work of Sims with yeast (7) and Cocking with
barley (7) has also shown that after giving N15 ammonia for relatively short periods (16 min & 2 hrs,
resp.) glutamic aci(d has the highest N15 content.
Yemnm and Folkes (24) state that in yeast, over 90 %
of ammonia may be assimilated by the glutamic acid
pathway. This conclusion is particularly interesting
since Roine (18) had observedl a rapid rise in alanine
after giving ammonia to nitrogen-deficient yeast.
In the Chlorella experiments reported here it was
found that the N'5 content of glutamic acid 'was considerably higher than the N15 content of alanine after
the administrationi of labeled ammonia. Assuming a
negligible metabolism of alanine in 1 minute of ammonia feeding, it cain be calculated that the 6.5 atom %
excess of N15 in alanine must have derived from a
compound with about 16.0 % N15. This suggests
that the alanine nitrogen dlerives from glutamic acid
(since transamination from glutamine to pyruvate
could Ilot be demonistratedl). This conclusion agrees
with the inability to (lenonstrate any mechanism for
the direct formation of alanine from ammonia. The
N15 data show that even if there were direct pathways
of alanine formiation they are not significant under
these experimental conditions and could not account
for the alanine increase after ammonia feeding.
Furthermore, the presence of an active glutamic acid
dehydrogenase andl glutamic acid-alanine transaminase
in Chlorella shows that these enzymes couldlprovi(le
the pathway) for alanine fornmation.
SUMMARY
The mechanism of the alanine increase in nitrogenstarved Chlorella following the addition of ammonium
chloride was investigated. Within 2 minutes alanine
showed a large rise. Pyruvic acid increased 50 %
in 5 minutes and a-ketoglutaric acid increased to a
lesser extent in 15 minutes. Adding pyruvic acid to
a Chlorella suspension resulted in a small increase
in alainine and a small decrease in glutamic acid. Dinitrophenol and deoxypyridoxine had little effect on
glutamic acid and alanine levels.
After feeding N15H4NO, to Chlorella for 1 and
5 minutes the amidle group of glutamine showed the
greatest N15 enrichment. The amino group of glutamine and glutamic acid had the next highest N15
content while that of alanine was lower. These re-
OF
N-STARVED
CELLS
211
sults are consistent with incorporation of (ammonia into organic combination catalyze(d by glutamic dehydrogenase and glutamine sy-nthetase. These enzymes
and glutamic acid-alanine transamiiinase are present in
Chlorella. Glutamiinie-alaninle transaminase and enzymes for direct amination of pyruvic acid were not
found. An amnmolnia-indluce(d increase in pyruvic acid
may be partially responsible for the increase of alanine, which occurs directly after adcministration of
amnmonlia to the low-nitrogen cells.
Ac K NO\VLEDGM EN TS
Mrs. Rose K. Gering ren(leredl able assistance in
much of this work. \We are indebted to Dr. R. J.
Volk of the Department of Soils at North Carolina
State College, Raleiglh, for the N15 analyses. We
also appreciate helpful coimments fromi Dr. S. I. Honda
and his colntinue(d interest throughout the course of
this work.
LITERATURE CITED
1. BULE:N, W.T A. 1956. The isolation & characterization of glutamic dehydrogenase from corn leaves.
Arch. Biochem. Bioplhys. 62: 173-183.
2. CHA-MBERS, R. W., J. G. MOFFATT, & H. G.
KHORANA. 1957. The preparation of nucleoside
5-phosphoramidates & the specific synthesis of
nucleotide coenzymes. J. Am. Chem. Soc. 79:
4240-4241.
3. CHIBNALL, A. C. 1939. Protein metabolism in the
plant. Yale University Press, New Haven. 306 pp.
4. CONNELL, G. E., G. H. DIXON, & C. S. HANES.
1955. Quantitative chromatographic methods for
the study of enzymic transpeptidation reactions.
Can. J. Biochem. Physiol. 33: 416-427.
5. DICKENS,vs F. & D. H. WILLIAMISON. 1958. The
preparation & properties of lithiurnt hydroxypyruvate and hydroxypyruvic acid. Biochem. J. 68:
74-81.
6. ELLIOT, WV. H. 1953. Isolationl of glutaminie synthetase & glutamyl-transferase from green peas.
J. Biol. Chem. 201: 661-672.
7. FOLKES, B. F. 1959. The position of amino acids
in the assimilationi of nitrogen & the synthesis of
proteins in plants. Symposium of Society for Exp.
Biol. 13: 126-147.
8. ISHERWOOD, F. A. & C. A. NIAVIS. 1956. Estimation of a keto acids in plant tissue: A critical study
of various methods of extraction as applied to
strawberry leaves, washed potato slices, & peas.
Biochem. J. 64: 549-558.
9. KATUNUMA, N. 1958. Adenylamidate as active intermediate in the fixation of the amino group of
amino acids. Arch. Biochem. Biophys. 76: 547548.
10. KRUPKA, R. M. & G. H. N. TOWERS. 1958. Studies
of the keto acids of wheat. 1. Behavior during
growth. Can. J. Botan. 36: 165-177.
11. LooAiis, W. D. 1958. The synthesis of amino acids.
In: Encyclopedia of Plant Physiology, W. Ruhland,
ed. Springer-Verlag, Berlin. 8: 224-248.
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212
PLANT PHYSIOLOGY
12. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, &
ROSE J. RANDALL. 1951. Protein measurement
with the Folin phenol reagent. J. Biol. Chem.
193: 265-275.
13. MACVICAR, R. & R. H. BURRIS. 1948. Studies on
nitrogen metabolism in tomato with use of isotopically labeled ammoniumiii sulfate. J. Biol. Chem.
176: 511-516.
14. MILLBANK, J. M/. 1953. Demonstration of transaminase systems in the alga Chlorella. Nature
171: 476-477.
15. MILLBANK, J. W. 1957. Keto-acids in the alga
Chlorella. Ann. Botan. 81: 23-31.
16. MYERS, J. 1946. Culture conditions & the development of the photosynthetic mechanism. III. Influence of light intensity onl cellular characteristics
of Chlorella. J. Gen. Physiol. 29: 419-427.
17. REISNER, G. S., ROSE K. GERING, & J. F. THOMPSON.
1960. Metabolism of nitrate & ammonia by
Chlorella. Plant Physiol. 35: 47-52.
18. RoINE, P. 1947. On the formation of primary
amino acids in the protein synthesis in yeast. Ann.
Acad. Sci. Fennicae Ser. A. II 26: 1-81.
19. THOMPSON, J. F. & C. J. MORRIS. 1959. The determination of amino acids from plants by paper
chromatography. Anal. Chem. 31: 1031-1037.
20. THOMPSON, J. F., C. J. MORRIS, & ROSE K. GERING.
1959. Purification of plant amino acids for paper
chromatography. Anal. Chem. 31: 1028-1031.
21. VICKERY, H. B., G. W. PI-CHER, R. SCHOENHEIMER,
& D. RITTENBERG. 1940. The assimilation of
ammonia niitrogen by the tobacco plant. A preliminiary study with isotopic nitrogen. J. Biol.
Chenm. 135: 531-539.
22. WIAMF, J. M. & A. PIERARD. 1956. Occurrence of
an L (+) alanine-dehydrogenase in Bacilluts subtilis. Nature 176: 1073-1075.
23. WILLIAMIS, R. J., R. E. EAKIN, E. BEERSTECHER, JR.,
& W. SHIVE. 1950. The biochemistry of B vitamins, Reinhold Publishing Co., New York. 741 pp.
24. YEMMi, E. W. & B. F. FOLKES. 1958. The metabolism of amino acids and proteins in plants. Ann.
Rev. Plant Physiol. 9: 245-280.
25. YEMAM, E. W. & A. J. \VrILLIS. 1956. The respirationi of barley plants. IX. The metabolism of
rioots during the assimilation of nitrogen. New
Phytol. 55: 229-252.
EXTRACTABILITY OF DNA & ITS DETERM\INATION
IN TISSUES OF HIGHER PLANTS 1 2
SIRKKA, KUPILA, 3 A. M. BRYAN, & HERBERT STERN
RESEARCH BRANCH, CANADIAN DEPARTMENT OF AGRICULTURE
INTRODUCTION
A number of methods have been describedI for (letermining deoxyribonucleic acid (DNA) in higher
plants. Three of these are commonly used: cold and
hot perchloric acid extraction (Ogur & Rosen, 5), hot
salt extraction (Martin & Morton, 4), and alkali extraction followed by acid hydrolysis (Schmidt &
Thannhauser, 6). In some procedures, DNA is first
separated from ribonucleic acid (RNA), in others
1 Received September 26, 1960.
Contribution No. 119. Plant Research Institute, Ottawa.
3 Post-doctoral fellow, National Research Council of
Canada. Present address: Botany Department, University of Turku, Turku, Finland.
4 Visiting Research Associate from Botany Department, University of Pennsylvania. Present address:
Morgan State College, Baltimore, Md.
5 Present address: Botany Department, University of
Illinois, Urbana.
2
not; in botlh cases, the analysis of choice has been the
measurement of deoxyribose content by means of the
diphenylamine reagent (1). Ultra-violet absorption
or phosplhorous content have been used instead of the
specific sugar reagent, but in general, these remain
unreliable indicators of DNA concentration.
Interests in some developmental problems such as
cell-division, root differentiation, and tumor formation
had led us to examine the relative merits of the above
procedures with a view to selecting a fully reliable
one. This has only been partly accomplished; differences between tissues prohibit a sinigle formulation.
METHODS
The procedures used in determining the DNA content of plant tissues may be divided into three parts:
I: Preparation of finely disintegrated fat-free powders; II: Extraction of DNA by means of a suitable
solvent; III: Determination of DNA concentration in
the extract by an appropriate analytical technique.
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