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L I B R A R Y
Michigan Sta tc
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ABSTRACT
NUCLEIC ACID METABOLISM IN YEAST: (l) STUDIES
ON TRANSFORWATICN OF THE ADENINE LOCUS BY CELL
EXTRaCTS; (2) STUDIES ON “DENOSINE DIPHOSPHaTE SULFURYLASE.

By Stuart William Bradford

1. EXperiments were performed which were designed to determine
if extracts of a yeast mutant could be used to alter the hereditary charac—
teristics of the nrototronh yeast. Cell free extracts of an adenine
red prototrODh cells. Such a treatment resulted in an increase in the
number of reversions to white, adenine indenendent cells. The results,
although preliminary, suggest that the mutants at the adenine locus in
.§aggharqmyces provide a system which can be conveniently utilized in
further studies concerned with establishing the existence of yeast trans-
formations. The alteration of heretable characteristics of bacteria by
essentially pure deoxyribonucleic acid (1) is well known and the mechanisms
involved have been studied. Transformations have not, however, been con-
clusively demonstrated in other organisms. Yeasts are genetically among
the most thoroughly investigated microorganisms and studies on the feasi-
bility of reast transformations are, consequently, of particular interest.

II. The nhosphorolysis of adenosine—S'-nhosnhosulfate (APS) by
inorganic ohosphate to form adenosine diphosphate and sulfate, which is
catalyzed by the enzyme ADP sulfurylase, has been reported (2). measure—
ment of this reaction in the reverse direction was investigated by substic
tution of molyhdate ion, an ion which has been shown to act as an analog
for sulfate in reactions catalyzed by adenosine trinhosnhate sulfurylase
(3), for sulfate. The back reaction, which is in the direction of aPS

formation, could serve as an alternative to the adenosine trinhosnhate

Stuart W. Pradford
sulfurylase catalyzed reaction, as a means of APS synthesis. Vith molyb-
date ion the reaction could not he demonstrated to proceed in the direction
of aPS synthesis, indicating that either the reaction has such a high
equilibrium constant as to be effectively irreversible or that in the “DP
sulfurylase reaction molybdate ion cannot substitute for sulfate.

A divalent zinc activated inorganic pyrophosphatase was observed
in yeast extracts. This enzyme may be identical to the zinc activated
yeast perphosphatase recently reported on (4). Earlier reports on yeast

oyrophosphatase had implicated only magnesium ion as an effective activator.

REFERENCES

 

l. Avery, O.T., C.M. MacLeod, and M. McCarty. 1944. Studies on the
chemical nature of the substance inducing transformation of
pneumococcal types. J. of Exp. Med. 22: 137-58.

2. Robbins, P.W. and F. Lipmann. 1959. Separation of the two enzymatic
phases in active sulfate synthesis. J. Biol. Chem. 222: 691-695.

3. Wilson, L.G. and R.S. Bandurski. 1958. Enzymatic reactions involving
sulfate. sulfite, selenate, and molybdate. J. Biol. Chem. 232: 975—81.

4. Schlesinger, M.J. and M.J. Coon. 1960. Hydrolysis of nucleoside di-
and triphosphates by crystalline preparations of yeast inorganic
pyrophosphatase. Biochem. BiOphy. acta..4l: 30-36.

NUCLEIC aCID KETaBCLISM IN YEAST: (1) STUDIES
ON TRaNSFCRMnTION or THE ADENINE LOCUS BY CELL
axraacrs; (2) STUDIES CN aDENCSINE DIPHOSPHATE SULFURYLaSE.

By
STUART WILLIAM BRADFORD

A THESIS

Submitted to
Michigan State University
in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Potany and Plant Pathology

1961

ACKNOWLEDGMENT

The author would like to express his sincere appreciation to
Dr. Robert Bandurski for his encouragement and help. The interest and
many helpful suggestions of Dr. alexander Kivlaan and Dr. Lloyd Wilson
are also gratefully acknowledged. The work reported on in this thesis
was supported by a National Science Foundation grant to Dr. Robert
Bandurski and by the Michigan Agricultural Experiment Station. Grateful
appreciation is also expressed to Dr. H. Roman for supplying the yeast

strains here employed.

ii

PaRT I:

R)

3.

A.

PART II:

TaBLE OF CONTENTS
STUDIES ON TRANSFORMATION OF THE ADENINF
LOCUS BY CELL EXTRACTS . . . . . . . . . . . .
INTRODUCTIO “ND REVIE"' CF LIT.an'L“RE . . . .

n. Genetic Transformation in Bacteria . . . .

 

 

B. The adenine Dependent Loci in Saccheromyces
C. Decxyribcnucleic acid in Yeast . . . . . .

mTERInLS aICD: T‘T" :iODS . . . . . . . . . .
3. Yeast Str;ins and Culture Kethods . . .

B. Preparation of ad(-) uC9t0n9 Powder . . .

C.‘1ethod of De ete rmini.u activity of the acetc.
PC-'-:-3-1r o o o o o . a a a o o o o a o o ,
D. Etfi rac ti and Determination of Yeast
‘TCIJ 1:313 hCl‘fiS o o o o o o o - O 0 O o a -

233:.LTS ' D o O O O O O O O a D F I o
n. Y3}: t Tr:“s-ormation Studies . . . . . .

B. Nucleic acid Extraction . . . . . . . . .

DISCTw‘SSICrI O O O O O O P O O O ‘ ‘ O O O ' .

STUDIES ON ADENOSINE DIPHOSPHATE SULFURYLASE .

INTRODUCTION a"D R"VIE" CF IITE: 7nTTRE . . . .

a. he Fcrmation of acid Adenylates . . . .
(1.) Sulfuric acid adenglates . . . . . . .
(2.) Biosynthetic reactions involving aDP

(3.) Sulfurylase reactions with molyhdate
B. Inorganic Pyrophosnhatase . . . . . . . . .
anTERIhLS L1- ‘jD T'HT: iODS r o o o o o o o a o o 0

RESULT

(I)

a. Release of Phosphate from nDP . . . . . .

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B. Divalent Zinc activation of

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4. DISCUSSION

SUMMARY .

REFERENCES .

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PART I: STUDIES ON TRANSFORMATION OF THE
ADENINE LOCUS BI CELL EXTRACTS
1. INTRODUCTION AND REVIEW OF LITERATURE
Genetic transformation by cell free extracts has only been demon-
strated to occur in bacteria; nevertheless, it seems reasonable to believe
that it may be possible to extend it to other microorganisms. In this thesis

the mutants at the adenine locus in the yeast genus Saccharomyceg were

 

utilized to study the possibility of transformation of yeasts. Extension

of the transformation phenomenon to include yeastswould provide a genetic
operation of great value. YeastSpossess a number of characteristics which
are advantageous for fundamental genetic studies. Many genera grow equally
well as haploids or diploids, single spores can be isolated and hybrids

rapidly produced, and finally tetrad analysis can be performed.

A. Genetic Transformatignflin Bactergg
Bacterial transformations may be simply represented as:
DNAl-X + bacterium-Y-————a>bacterium—X
where bacterium-X possesses heritable characteristics not possessed by Y,
and DNA-X is DNA extracted from cells of bacteria-X. In general, transforma-
tions are reciprocal and
DNA—Y + bacterium-X-—————’tbacterium-Y
may occur. This phenomenon, whose dependency on DNA was discovered by
Avery, MacLeod, and McCarty for Diplococcusgpneumoniae in 1944 (1), has
since been extended to a number of other species of bacteria. Transforma-
tions have been reported in Neisseria meingitidis (2), flgggphilggfiinflugggge (3).

1The following abbreviations are used: RNA, polyribonucleic
acid; DNA, polydeoxyribonucleic acid; Tris, tris (hydroxylmethyl) amino—
methane; TCA, trichloroacetic acid.

Xanthomonas phaseoli (A), Salmggglla_typhemurim (5), Rhizohium meliloti (6).

 

 

and Bacillus sphtilis (7). The reported transformations encompass a variety
of characteristics: antigenic specificity, drug resistance, cellular and
colonial morphology, and the ability to utilize particular nutrients. In
addition, transformations between bacteria regarded as separate species
have been reported (8,9). Although the work of Avery and his collaborators
was performed with highly purified DNA, in work by subsequent experimenters
transformations have often been performed with crudcr preparations (4,5,7)
but in all cases the ability to initiate transformation was reported to

be DNase sensitive.

Studies on transformation have been pursued most thoroughly in

Hemophilus influenzae and Pneumococcus. In Pneumococcgs transformations

 

do not occur unless bovine serum albumin and calcium ion are present (1).
The exact role that these two factors play is unknown, but it is very likely
a membrane permeability effect.

In H. influenza; transformations can take place in simple saline

 

solution (10). In other species the conditions which might be necessary
have not been established. For example, in Xanthomonas phaseoli. in which
transformations were affected with a crude extract, removal of protein from
the active nucleic acid preparation increased the rate of transformation
10--lOO fold, and addition of 0.01% commercial yeast extract increased the
rate as much as 10,000 times (4)1

The stability of H. influenzae transforming DNA under a variety

 

of physical and chemical conditions has been investigated (11). No loss
of activity was reported to occur after heating for one hour at 81° in

citrate buffer or from treatment of the DNA with solutions of pH 5.0 to
10.5, but about 80% of the activity was lost in a solution of pH 3.5 or

in a solution of pH 11.0. DNA solutions in distilled water were quickly

inactivated and highly purified preparations lost activity when vacuum
dried. Litt (12) has shown in Egeumgggcgus that mechanical breakage of

DNA molecules diminishes the number of transformations obtained. Insta-
bility on freezing and thawing has been reported for a crude preparation
of transforming DNA (A). Marmur and Doty (13), working with Qiplggggggg
lpgggmggiag, have concluded that the double helix of DNA is necessary for
transforming ability. In heated solutions of DNA the strands of the helix
separate and transforming activity is lost, while upon slow cooling the
strands reunite and regain the ability to transform. Hybrid helices, which
are reconstituted between transforming and non-transforming DNA, also possess
transforming activity.

It is now well established that a single DNA molecule is suffi-
cient to effect a transformation, and, therefore, there is a linear relation-
ship between the concentration of transforming DNA to which the cells are
exposed and the number of transformed cells obtained (14). Further studies
have demonstrated that a single DNA molecule may transform more than one
characteristic of a cell. This is illustrated by the mannitol utilization
(I) and streptomycin resistance (8) mutations in figegmgggggug (15). DNA
extracted from the double mutant (presumably MS - DNA), transforms a much
higher proportion of wild type cells into MS-cells than are transformed
by a mixture of DNA obtained from the single mutants (M—DNA plus S~DNa).

The mechanisms involved in the genetic conversion of cells by
DNA hawareceived considerable study and it is now known that transformation
involves a series of steps. First, the bacterium must be physiologically
competent to undergo transformation. This receptive state, although
occurring at random in normal growing cultures, may be made to appear

cyclically by brief eXposure of the culture to a low temperature (16).

A

Fox and Hotchkiss (17) have shown that the develOpment of competence con—
sists in the appearance of sites with which the transforming DNA combines
reversibly. Populations of competent cells were frozen in 10% glycerol.
Under these conditions the cells retained their competence and could be
thawed, treated with DNA under a variety of conditions and the reaction
terminated by DNase. At low concentration of DNA the response obtained

was linear, but at higher concentration DNA became saturating and the number
of transformed cells obtained did not increase. Such a relationship can

be treated according to conventional enzyme kinetics:

k1 1‘3
8(3) + DNA? 3(3) + DNA complex ———-—~—>B(tr)

where 3(5) represents bacterial sites, and B(tr) represents transformed
bacteria. Hotchkiss' and Fox's calculations indicate between 33 and 75
sites per cell. The actual nature of the sites is unknown; however, several
observations suggest that enzymatic processes are involved: their formation
is stimulated by an apparent protein synthesizing system (17); the absorp-
tion of DNA onto the sites is a temperature dependent reaction (18), and
they react with some DNA more readily than others, thus exhibiting a speci-
ficity (19). In addition, Schaeffer (9) has shown that the sites must be

on the surface of the bacterium.

Following adsorption onto a site, the transforming DNA is retained
in a DNase resistant form, presumably representing penetration into the
cell. The use of P32 labeled DNA has established that the number of trans-
formed cells obtained is directly related to the amount of DNA which is
permanently bound by the cells (19). Non—transforming DNA can also be
taken up and Lerman and Tolmach (18) have shown in Egggmogqqggg that
.§e~92li DNA will accumulate to the same extent as the genetically active

DNA extracted from Egegmngqcus itself. However, when several species of

5
bacteria which have not been transformed were exposed to.§£§2fl9€9€9§§ DNA,
no uptake of DNA occurred. In view of this, the authors suggested that
measurement of the incorporation of labeled DNA into microorganisms might
prove to be valuable in screening for these organisms in which transforma-
tion is feasible.

The actual amount of DNA taken up by receptive cells can be in-
hibited by addition of foreign DNA - non-transforming DNA from the same
species, or even DNA from other species; but the degree of inhibition varies
with the particular DNA employed. DNA from more closely related species
inhibits more, while that from completely unrelated organisms, such as the
DNA from calf thymus glands, is much less effective (20). The number of
DNA molecules incorporated for each cell transformed, in the case of strep-
tomycin resistance in Hemoohilug, was found to be 120 (19). This, in view
of the fact that one DNA molecule is believed to be sufficient to effect
a transformation, strongly suggests that for figmgphilgs one out of every
120 DNA molecules carries the gene for streptomycin resistance.

The occurrence of reciprocal transformations almost certainly
establishes that transforming DNA is not merely carried along, but, on the
contrary, becomes eventually incorporated into the "chromosomal” material
of the recipient bacterium. This incorporation must logically be preceded
by a pairing of the exogenous DNA with some complementary site on a chromo-
somal structure of the bacterium. The necessity for such specific pairing
or synapsis is indicated by Schaeffer's (9) studies on transformation to
streptomycin resistance performed between separate species of bacteria.
Such interspecific transformations occur with a much lower frequency than
those within a Species, although there is no reduction in uptake of the
transforming DNA. This situation may be interpreted as follows: in

intergpggifig transformations structural differences around the streptomycin

6
region act as obstacles to pairing and thereby reduce the frequency of
structurally similar to the DNA of the recipient bacteria and pairing may
occur more easily.

The final step in the series of events leading to a transformed
cell is the incorporation (or copying) of the genetic factors, brought
within the cell by the DNA molecule, into a chromosomal structure. Hotchkiss
(21) has described a complex single locus which he designates 33.322, con-
veying a high level of streptomycin resistance. A molecule of transforming
DNA carrying the abd.complement may transform a recipient cell to any one
of the different characteristic low levels of resistance corresponding to
the g, b, orIQ sites on the complex locus or less frequently to the charac-
teristic higher levels corresponding to the 2b, pg, or 39d parts of the
complex locus. However, ifIQ is incorporated into the recipient cell then
the pg sites are not given phenotypic expressions; similarly, if 3b is in-
corporated into the recipient cell thenIQ is not expressed. Therefore only
a portion of DNA molecule is incorporated, or copied, by a cell undergoing
transformation and the genetic markers on the remaining portion of the
molecule appear to exert no influence. The incorporation of the transform-
ing DNA apparently does not occur until after several cellular divisions,
because transformed cells give rise, for the first few divisions, to both
normal cells and transformed cells.

A few accounts have appeared of the altering of heritable charac-
teristics in bacteria by exposure to suitable RNA preparations. Kramer
and Straub (22) were able to induce the formation of penicillinase in strains

of gacillus‘gerus lacking the enzyme by extracts from a penicillinase pro-

 

ducing strain. The time required for the formation of this induced enzyme

was considerably reduced by treatment with RNase, suggesting that the active

 

   
  
  
  
  
  
   
 
 
    
 
  

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moieties were low molecular weight noly— or oligonucleotides. In a similar
manner Reiner and Goodman (23) found that formation of the induced enzyme
glucokinase in'§;~qgli could be accelerated by the dialyzable fragments of
RNase treated RNA from a strain of ggflggli producing glucokinase. These
RNA mediated "transformations" appear to bear little relationship to the
DNA transformations, since the activity of the RNA is not lost upon depoly-

merization.

 

B. .Ihg.¢Qggigg‘QgEQQQent ngi_in Saccharomngg

Roman has studied mutations to adenine dependence in §ag__ _ myggs
(24) and describes recessive mutations at seven loci. At two of the loci
the mutants form a red pigment when supplied with adenine, but the mutants
at the remaining five loci, although adenine requiring, remain normal

colored. These seven loci are shown below in relationship to the formation

of red pigment and adenine (the mutant adg also requires histidine for

 

 

 

growth):
ad3 ad ad5 ado ad7 ail ad2
i X \ T > 4" T i) adenine
red
histidine pigment

.ggg and gdz are tightly linked, but at the remaining loci the genes exhibit
regular Mendelian inheritance and segregate independently. 'gdl, Egg, £12
and age are leaky mutants; i.e., they allow some small amount of growth
in the absence of adenine, and form pink colonies.

Evidence has been provided by Roman for the occurrence of crossing
over during mitotic division in the diploid strains of the ad mutants.
When two haploid strains, containing mutation of different origin but of
the same locus, are crossed to provide diploids, reversions to adenine

independence occurs with a frequency of .01% to .0001%. For example,

8

mutant strain adR-l crossed with mutant strain 223:2 gave a small number
of adenine independent reversions. If Eégfil and ad2-2 represent different
defective parts in the same locus then crossing over between these two
points would restore the normal adenine independent gene. Analysis of the
revertant diploid showed that crossing-over had occurred, although in a
few cases non—reciprocal recombination was demonstrated. By use of this
technique the various.gd loci were found to each contain from A to 29
separable alleles. However, these represent minima and additional alleles
will probably be disclosed as more mutants are studied.

Similar adenine requiring mutants occur in Neurospoga and have
been utilized to formulate some of the metabolic pathways involved in

adenine synthesis:

 

 

glycine
acetate 1) «£>inosine ;:adenosine
C02 ’
m, L If
pigment adenine
accumulation

in which the diagonal lines represent different mutants. The red pigment
is a mixture of water soluble polymers related-in structure to amino imidzaole
carboxamide. This pigment or some of its precursors are slightly inhibitory
to growth and cultures of the red mutant have been observed to become white
but still adenine requiring owing to selection for a second mutant prevent—
ing the formation of the pigment (25). There is some question whether
adenine dependent mutants always represent a defect in biosynthetic pathway
leading to the production of adenine. Abram (26) has described a red adenine
requiring mutant in §§gghargmygg§, which is capable of synthesizing adenine
at a rate only slightly less than that exhibited in the wild type.

Red strains of Saccharomyces are known to be unstable and, as

would be expected, the instability is most pronounced in the haploids (27).

Ills.”

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On solid medium haploid colonies produce white papillae, while in liquid
medium the white variants rapidly outgrow their red progenitors. This
selection is very much more rapid in shaken cultures than in standing
cultures, with the exception of red respiratory mutants of the petite type
in which the selection pressure is the same under both conditions. The
cause for the Selection remains unknown: very little selection occurs
under anaerobic conditions and very little red pigment is produced under
these conditions. Therefore, it is likely that either the pigment itself
or some precursor has a deleterious effect on the growth of the red strains
thus allowing selection for a mutant which inhibits pigment production
(28). Srb (29) has also noted that there is a strong selection for the

white prototroph when red strains are grown at a pH below 5.

C. Deogyribonucleic acid in Yeasts

In yeaststhe amount of DNA is greatly exceeded by the amount
of RNA. RNA composes about 95% of their total nucleic acid and conse-
quently the extraction and isolation of yeast DNA poses special problems.
Chargaff has given the only report of isolation of yeast DNA. He extracted
with l N NaCl for 72 hours, and removed RNA by a combination of precipita-
tion with calcium ion and dialysis with RNase (30).

A general review of methods for isolation of DNA appears in the
volume edited by Chargaff and Davidson (31). In general, the isolation
methods involve extraction with l N NaCl or with detergents. Extraction
with cold 20% alcohol (32) and with the aid of cetyl trimethylammonium
bromide (Cetavlon) have also been reported (33). Removal of protein is
usually accomplished by shaking with a mixture of chloroform and octonal,
but dialysis with chymotrypsin may be used and neither of these methods

appears to degrade DNA (34). The removal of contaminating RNA is often

lO

difficult, and although RNA

I)

e can he used, it leaves an enzyme resistant
polynucleotide core. Adsorption of DNn on charcoal (35) or precipitation of

DNA by cetyl—trimethylarmonium bromide tum; proved effective in some

0

ases.
A yeast DNase has been described by Chargaf and Zamenhof (36).
The enzyme is released only gradually from yeast extracts and its appear-
ance is controlled by the following sequence of events: upon standing a
proteinase is released from the extract which digests an inhibitor of the

enzyme resulting in the release of active DNase.

2. MATERIALS AND METHODS
A . 12812391111. -5. :1. ii -Qeiilizej‘ietheis

A red, adenine requiring, diploid strain of
supplied by Dr. Herschel L. Roman (Roman #092-0-106). The white, adenine
independent wild type was obtained as a back mutant from this strain. In
this study the two strains are designated as ad(+) and ad(-), respectively.
The ad(+) and ad(-) mutants may be considered to differ in only one gene
since adenine dependence and red color result from a recessive mutation
at a single locus (¢A)o

‘Iypes-q§;quia. The following types of media were employed:

I§Qz a synthetic medium containing the following ingredients per liter

of solution:

amronium sulfate 1 g thiamine-HCL 40004”?
glucose 20 g FeCl3’6 H2O 500,ug
histidine-HCL 10 mg ZnSOL-VHQO 700,pg
methionine 10 mg H3803 100 ’ug
tryptophane 10 mg CuSO4’5H20 1% lug
uracil 10 mg KI 100 pg
adenine sulfate 10 mg KHQPOA 975 mg
biotin 20,0g KHPOA 125 mg
inositol 20 mg MgSOA'7H20 500 mg
calcium pantothenate 4000,2g NaCl 130 mg
pyridoxine—HCL A000,;g CaClz'2H20 100 mg

15 grams of agar were added to prepare solid media.

11

Egg: 30 minus adenine.

§;29: A complex medium containing 2.5% glucose, 0.3% bactopeptone,
0.2% bacto~malt extract and the same concentrations of all the minerals
used in SC medium with the exception that no NHASOA was added. 1.5% agar
was added to prepare solid medium.

In all cases the media were autoclaved for 20 to 30 minutes,

the exact time depending on the volume being sterilized. The pH before

autoclaving of the SC medium was 6.0 and the pH for the 0-30 medium was 5.9.

 

Maintenangg of cultures. Cultures were maintained by weekly sub-
culture on 0-30 slants. Red ad(+) cells became visibly pigmented sooner
on C-30 medium than on WAD medium but after 4 or 5 days both were equally
dark red. White mutant colonies appearedyfrequently on ad(+) slants and
in 2 or 3 weeks, upon solid medium; or in 3 or 4 days, in the case of
populations transferred daily in liquid medium, completely outgrow their

red progenitors. Therefore ad(+) slants were always inoculated with red

colonies appearing from single cell isolates of the ad(+) strain.

B. Preparation of ad(-) Acetone Powdgg

The ad(-) cells were grown in 0-30 medium in shaken Erlenmeyer
flasks at 30°C. The cells were harvested by centrifugation, washed with
pH 7.5 M/lO sodium citrate followed by distilled water. They were then
dispersed in a few ml of distilled water, mixed well with 150 ml of -20°
acetone in a high speed mixer and dried in a sterile tube under vacuum.
From 600 ml of medium containing 3 x 1010 cells, 510 mg of very fine white
powder was obtained. This powder, although free of bacterial and fungal
contamination, still contained an average of 10 viable yeast cells per mg
Of acetone powder. Since neither a second cold acetone treatment nor ex-

posure of the powder to the fumes of propylene oxide in an evacuated

12

desiccator served to decrease this number, the powder was completely
sterilized before each experiment by addition oft“.5 ml of cold 95% ethanol
for each mg of acetone powder contained in a sterile tube; after 30 minutes
at 10 this mixture was diluted with sufficient WAD medium to bring the

concentration of powder to 1 mg per ml.

 

C. Method of Determining actiy}jy;g§_the‘2§etone Powder

 

The ad(+) cultures from a sufficient number of 6 to 8 day old
0—30 slants were suspended in WAD medium, cell counts made with a Neubauer
hemocytometen and the cell suspensions diluted to the desired density with
additional WAD medium. 20 ml of WAD medium containing 1 mg per ml of
ethanol sterilized acetone powder was mixed with 20 ml of cell suSpension
in a 250 ml erlenmeyer flask (or half these quantities in a 125 ml flask)
and shaken at 30° for 8 hours. at the same time 20 m1 of cell suspension
was shaken with 20 ml of WAD medium containing an amount of alcohol equal
to that found in the acetone powder suSpension. At the end of the period
of shaking, aliquots were withdrawn and pipetted into 41° WAD agar in the
amount of 1 ml for every 19 m1 of medium. The cells were mixed thoroughly
throughout the warm agar by means of an enclosed magnetic stirrer and the
agar poured in the amount of approximately 20 ml per petri plate. Although
the amount of medium poured and thus the number of cells per plate was not
controlled, a measured total volume of agar was poured in each experiment;
and, therefore, the total number of cells plated was always known.

For studying the effect of anaerobic conditions on colony growth
appropriate numbers of cells were spread on the surface of WC agar by
tilting and rotating the plates and after 4 hours incubation were covered

with a 1-2 mm layer of 41°C 70 agar.

l3

Napleig 9. La

0- giresfloa.apflfizaipeflpn.sfleeei- - i -
Twenty-five grams cf acetone powder of baker's yeast was
stirred for 20 minutes at 50-1000 with a high speed mixer at 10,000 r.p.m.
in 100 ml of water containing the following: 0.1 M NaCl, 0.01 M pH 9.0
Tris-H01, 0.5 mm EDTA, 1 gram sodium dodecyl sulfate, and 50 grams 200/1
glass beads. The resulting mixture was decanted, centrifuged at 10,000
x g for 10 minutes, the supernatant precipitated with 2 volumes of cold
100% ethanol and centrifuged at 22,000 x g for 15 minutes. The alcohol
precipitate was taken up in 50 ml 1 M NaCl containing .01 M pH Q.O Tris-
HCl, and .05 M EDTA, and freed of protein by stirring in the high speed
mixer with an equal volume of 9:1 chloroform-octonal, centrifuging at 2500
r.p.m., and removing the upper aqueous layer. This process was repeated
with 0.5 volume portions of chloroform-octonal mixture until no more protein-
aseous scum formed at the aqueous—organic interface; generally 4 additional
times were sufficient. All the chloroform-octonal mixture used in the
deproteinization was combined and re—extracted twice with 50 m1 of 1 M NaCl.
all the NaCl extracts were combined, precipitated with 2 volumes cold 100%
ethanol and centrifuged at 22,000 x g for 15 minutes. This residue could
be dissolved in l M NaCl, giving a solution in which DNa composed about
12% of the nucleic acid present, or dissolved in 15 ml of 10% Ca012 and
centrifuged at 22,000 x g for 50 minutes in which case 33% of the nucleic
acid in the supernatant fluid was DNfl.

Determination of nucleic acids. Samples were centrifuged at

-.‘-—.-‘----. 0-. .-Q

 

5000 r.p.m., extracted twice with 2-1/2 volumes of 10% TCA and twice with
2—1/2 volumes 1:1 ethanol-ethyl ether. The residue was hydrolyzed by
heating in a water bath at 90° for 15 min. centrifuged at 5000 r.p.m. and
tine supernatant fluid saved for the determination of RNA and DNA. RNa

was determined by the method of Dische (37) involving reaction with

14

concentrated H2804 and cysteine. DNA was determined by the diphenylamine

method of Dische (38).

3. RESULTS
A. Yeast Transformation Studies

The effects of shaking a suspension containing 1 x 10-6 ad(+)
cells per ml with and without the addition of acetone powder from ad(-)
cells aneshown in Table 1. The level of acetone powder employed, 1 mg
per ml, corresponds to 2 x 106 living ad(-) cells.

The diploid strain of yeast used in these studies is quite stable
and ad(+) slants containing as many as 75 x 106 cells may, upon plating,
give no adenine independent colonies. However, on occasional slants,
sectors of white revertant cells do occur. In preparation of suspensions of
red cells, slants which have visible growth of white cells are discarded, but
slants which may contain sub-visible white colonies cannot be eliminated. In
consequence, the number of white colonies obtained upon plating the suspension
represents the number of white cells and their progeny originally present on
the slants used plus whatever number of cells (and their progeny) which revert
to adenine independence during the period of shaking. Two hundred eighty-five
more colonies appear on plates obtained from the population shaken with
acetone powder, Table 1. In all 15 pairs of plates, the plates which repre-
sent cells treated with the acetone powder preparation have a larger number
of colonies. If all the additional colonies were the result of reversions
occurring within a short time following the mixing of the acetone powder
with the cell suspensions, then a reversion or transformation rate of 2.5
to 5.0 cells/106 cells originally present could account for the additional
colonies. If, on the other hand, the reversions occurred with equal fre-

quency throughout the period of shaking, then the approximate rate per

.‘. .4 - I<<

.nHHoo pOH x H hHopoEonuoam nuHs popstooan opmHQ some unopeoo
cocoons psospHe can 59H: coanm nHHoo 00H x mH .nopeoo ago no
mprHpoe manHenopop mo avenues pops: pothonop mm mQOHpHpcoo

 

 

m.oo mVOH we no we no mm mm cm N0 hm Hm be 0m «m on 00 +
m.0m wmb on we pm on me Nm mm mm Hm He He em mm on we u
mH «H MH NH HH OH 0 w b o m w m m H
noHGOHoo honesz opmHm nopsom
ouMHm pom opwng mo econoOd
.oz owmno>¢ .oz Hmpoa Atvpe

 

mcHumoQQ¢ noH:0Hoo opHnB mo .02

 

hopsom ocopooa Alvpe spH: uncapmoae

maHBOHHoa "HHoo paneaoaoeaH oaaaoea apnea op uHHoo
pom .mchHsaom qucop¢ mo aconpobom mo popasz

H quda

 

l5

generation time can be simply arrived at. Tith a generation time of 2
hours, the reversions occurring at times 0-2 hours would multiply to give

8 cells at the end of the shaking time, those at 2—4 hours would give 4,
4—6 hours would give 2, and 6-8 hours would give 1. The total number of
reversions or transformations obtained is equal to the above numbers summed
times the mutation rate per generation time (X). Therefore, 15X must equal
285 and X must equal approximately 20. Since 15 x 106 cells were used a

frequency of l-l/3:x 10"6

cells reverted or transformed per generation
time of 2 hours could account for the additional colonies obtained from
plates of the acetone treated cells.

The question arises as to whether the white colonies shown in
Table 1 represent, in actuality, ad(-) cells since the production of red
pigment by adenine dependent mutants is inhibited by growth under anaerobic
conditions. Furthermore, the addition of yeast acetone powder to the
adenine deficient medium might promote the growth of ad(-) cells. To
answer the first of these two questions, suitable dilutions of ad(-) cells
were grown on the surface of SC agar or between layers of SC agar. It was
shown, Table 2, that under these conditions the colonies growing under an
agar layer accumulate red pigment more slowly and never obtain so deep a
pigmentation as those grown aerobically. Nevertheless, the ad(—) colonies
do exhibit a light pink color after 10 days which is easily distinguishable
from the white colonies formed by ad(-) calls. all the white colonies
listed in Table I remained completely white for at least 3 weeks after
inoculation.

In regard to the second consideration, it was found that yeast
acetone powder does not serve to increase the rate of multiplication of
ad(+) cells, when they are grown in adenine deficient medium. Table 3

shows that even when ad(—) acetone powder is supplied at the rate of 1 mg -

TABLE 2

Effect of Aeration on Development of Red Color in Ad(—)
Colonies and upon the Number of Colonies Formed

 

 

 

Growth Average No. Color after Color after
conditions of colonies/ 5 days 10 days
plate
Aerobic 192 Pink Red
Anaerobic 188 White Light Pink.

 

 

Conditions as described under methods of determining
activity of acetone powder. Each an average of 4 plates.

TABLE 3

Effect of Ad(+) Acetone Powder on Growth
of Ad(-) Cells

 

Average No. of colonies/plate

Ad(—) cells shaken in:

 

 

0 hours 8 hours
WAD media 83 49
WAD media with 86 55

1 mg/ml Ad(+) acetone pwd.

Plated on SC media. Each an average of 5 plates.

 

f ‘w m.fl_«-f‘.——T— “—7—-

16

which represents the yield from about 2 x 106 ad(-) cells - for approxi«
mately every 100 ad(+) cells, there is no stimulatory effect on the growth
of these cells.

A considerable decrease in viable cell number occurs in popula-
tions of ad(+) cells following 8 hours of shaking in an adenineless medium,

Tables 1 and 3. Although the zero time cell counts, shown in Table l, were

'1“

made with a hemocytometer and therefore did not distinguish between living

Y

 

and dead cells, separate counts made both by dilution and colony counting
and by use of the hemocytometer established that all or practically all

cells on fresh ad(+) slants are living and viable.

A“ .Av-Jun’eu—‘u ‘.‘

‘0

B . 3119.19.13.39. enemies.

Because of the preponderance of RNA present in yeast (Table 4)
nucleic acid extractions were performed mainly with the view of increasing
DNA yield. For this reason one molar NaCl was employed, without regard
to the fact that RNA nucleOproteins are insoluble in this concentration
of NaCl. Preliminary eXperiments demonstrated that extractions of whole
cells or of acetone powder for 24 hours with molar NaCl resulted in a very
low yield - in the latter case only 4% of the total DNA was extracted.
(lrinding the acetone powder increased the yield several fold, while with
the procedure finally adopted, which utilized grinding in the presence of
sodium dodecyl sulfate, as much as 42% of the total DNA was extracted
(Table 4). However, this high yield occurred only from acetone powder #1,
‘xhereas subsequent extractions from acetone powder #2 gave a maximum of
only'lbleNA. although the basis for this difference was not established,
aa‘pOSSible explanation is suggested by the fact that powder #1 was made

fjxmn yeast that was frozen and thawed before treatments with cold acetone,

vfliile #2 represents fresh cells processed directly with acetone.

mhh_.
:1
.‘C
«
,
,
I

‘I’i’i’ll’ibil’

 

 

 

 

e.m «mm m.oH mumpaafioouq maumo .e
N.om em 0.0 scopmcpoaan Naomo .m
o.m b.0H m.oa mam o.vm endow ofimHosc ovumncfiououa Hapoa .v
m.m MHH 0.0 HocwpoOImHoom spat cadpoaupxo ccooom .m
<.¢ ow w.v HonopoOIMHoom spa: nofipooupxo unnfim .N
®.©H em N.oH g pneumaumasn.wouwcwopouaon .H
mm.b o.bH v.0 mam on oouuuxo Hhooooolwz .N .02 povsoa encased .HH
m.me N.OH “.0 owqa mo posupxo Hsooeoe-sz .H .02 season ocouooe .H
tun nun NH.m Omar Hmm unao< cacaosz Hence
. Y1; 11‘ 11 4 1‘ 11 ‘4 '1‘
.42a ezm uvfioe
Houoa m Hence m odoaosz Away Amsv cowvoouh
Haves :H 42m 4:9
muopooom u aza

 

 

 

pnmow n.noxmm scum nvdoe cacaosz mo coupomuuxm

w mqmda

4. DISCUSSION

The well established studies on the genetic role of DNA in
bacterial transformation suggested that it Would be worthwhile to seek a
similar system in yeasts. however, in view of the occasional reports of
transformation-like phenomena mediated by RNa, it seemed advisable to
investigate first the possible hereditary effects of crude, mixed nucleic
acid extracts on yeasts. A system has been described in this thesis,
utilizing an adenine mutant 0f.§E9QQi£QWXEE§: in which extracts having
possible transforming activity might be assayed relatively easily. nISO,
a procedure is outlined for obtaining DNA enriched nucleic acid extracts
from yeasts.

The preliminary results which have been reported here indicate
that yeast cells can be modified by exposure to cell-free extracts. This
transformation of adenine negative to adenine positive cells by unfraction-
ated acetone powders of the latter suggests that genetic transformation
may have occurred.

In experiments of this sort it is often possible to explain the
differential effect as being a matter of selection. Such an exnlanation
is particularly in order in the experiments reported on, where a control
of ad(+) cells treated with ad(+) acetone powder was not provided. (It
proved difficult to grow sufficient quantities of ad(+) to produce an
acetone powder, due to back mutation to, and selection for, the white
‘prototroph.) Thus the yeast acetone powder may be suggested to have had
:3 growth promoting effect on the ad(-) cells which were already present,
or which arose by mutation from ad{+) cells during the shaking period.
It; was demonstrated that the growth of ad(+) cells was not stimulated, in
cidenincless media, by high concentrations of ad(-) acetone powder. In

emienineless media viable ad(+) cells gradually decrease in number, and the

ll" - it“ In.--

I ITWST—fT__‘f.‘

18

addition of ad(-) acetone powder in a ratio of powder/living cells 10,000
times as high as that used in the transformation experiments does not
”act this decrease.
an effect of a non-specific factor on alteration of an inherited
characte istic in yeast he 5 been reported (39). Galactose—positive cells

were converted to galactose-negative cells by extracts not only from

l|

filactose positive cells, but from extracts of baker's yeast and even from
galactose negative cells.
Ashida, Minagawa, and co-:.orkers have studied extensively an

alteration in conper ion sensitivity of a Saccherom'ce llipsgi e 3 strain,

q o
.1 fl.
‘ “n-“‘ *‘-W~d‘- “.m-

1" —“-—-_—.—.f.— —.W

1

induced by a specifi 1c but not reproduced factor (40,41). I this yeast

is plated on a medium containing CuIQ, only 600 of the cells plated form
colonies; but resistant strains can be selected in which 100% of the cells
plated give colonies. RNA extracted from resistant strains can convey
resistance to the sensitive strains, while the correSponding extract from

sensitive cells does not. However, the effective RNa does not seem to be

reproduced in cells which are treated with a RNA extract (42).

{P

During the time that this thesis was bein ng writte paper appeared
by Oppenoarth (43), in ."hich he reported his experiments on ye trans-
formation. a crude nucleic acid extract from a yeast strain able to ferment

maltose, sucrose, and other dissacharides was added to cultures of a strain

nt dissacharides. The non-fermentors were left to grow

(0

not able to ferm
in the presence of the yeast extract for several days,then transferred to
fermentation tubes. The cells were now found to possess the ability to
ferment one or more diss acha rides. Some of the tran formed 3e.st retained
the acquired ability following sub-culturing 20 times, while other could
no longer ferment after Ming re—inoculated only a few times. On the has is

of experiments with added RNase, DNase, or proteinases, the author believed

19

that the permanent transformation resulted from ingestion of DH“, Thereas
the temporary or pseudo-transformations were RNa induced. The latter case

resembles the specific RNA induced Cu+2 resistance in Sacchazomygeg.

“~—

The Saccharonyces mutants at the adenine loci can be conveniently

-wc»

east extracts fcr genetic transforming

S:
(+
H-
1...;
Ho
{0
(D
{L
C13
U}
{U
3
(0
CD
:3
’0
’3
H)
B?
U)
a}
C3
'V‘!
F).
J
x '3
<4

activity. The adenine requiring yeast cells, which are red, may be mixed

fl!‘
1-

with extracts from the white, adenine independent cells, and plated on
adenineless media. Transformed cells will give rise to colonies on the
adenineless plates and, further, the colonies which represent transforma-

tions will appear as white and not red.

”'1‘ w: ‘lr'h‘m “Mia—‘3'
0

4
“A-

!

Genetically the adenine mutants have been rather thoroughly
investigated, and crossover frequencies between loci and within loci measured.
In addition, the metabolic pathways involved in red pigment formation and
adenine biosvnthesis have to some extent been established. In contrast,
in the copper resistant mutants of yeast (40), neither the genetics nor
the physiological processes involved are clear. In the dissacharide utilizing
mutants of yeast (43), quantitative analysis for transformations are diffi-
cult because fermentation measurements are required.

Extraction of nucleic acids from yeast cells proved difficult
arui detergent.extraction(fi‘acetone powders yielded less than 10% of the total
ruicleic acid present. Similar low yields were reported by Oppenoarth (43).
Pkmwever, he was later able to increase considerably the amount of nucleic
acixi obtained by using the gut juice 0f.§§li£.fl€fli§i§ to produce yeast

pxwytOplasts. From the nrotorlasts DNA and RNA could be extracted in high

J'iel'iso

PART II. STUDIES ON ADENOSINE DIPHOSPHATE SULFURYLASE

1. INTRODUCTION AND REVIEW OF LITERATURE

Mononucleotide acid anhydrides are usually biosynthesized by
the pyrophosphorolytic cleavage of a nucleoside triphosphate. Thus the
biosynthesis of the mixed acid anhydride adenosine-—%'-nhosnhosulfate.

fl
aPS‘, has been shown to be linked to the cleavage of pyrophosphate from

3‘"
ATP. In addition, however, Robbins and Lipmann (4A) have reported the E
phosphorolysis of APS with the resultant formation of ADP — in the reverse a

if
reaction phosphate would be cleaved from “DP and APS synthesized. Syn— 5

a

thesis of APS by this reaction mechanism therefore would represent an

iv

unusual mode of formation of nucleoside acid anhydride. The reaction is
of further, more general interest in that the energy used to drive the
reaction in the direction of aPS synthesis would have to be derived not
from the phosphate bond energy of nTP but from that of ADP.

It was the purpose of this study to measure the aDP sulfurylase
reaction in the direction of aPS synthesis. The enzymatic cleavage of Pi
from “DP in the present of M0022 was utilized as a means of assay for
the reaction in this direction. Molybdate ion has been demonstrated to

substitute for sulfate ion in reactions catalyzed by aTP sulfurylase (AS).

A . Eliefsmaiisn o 5‘ n C i '3 “Clematis

 

among the ménonucleotide acid anhydrides those of aMP (the
acid adenylates) participate most frequently in biochemical reactions.

In addition to the phosphoric or pyrophosphoric acid adenylates (aDP, aTP),

------ O- -a--.r--- - -- -r—QC-O“~H_-‘ H” C - C -7“-

2The following abtreviations are used: 5P8, adenosine—S'-
rhosphosulfate; PaPS, 3'—phosphoadenosine-5'phosphosulpate; PP, pyro—
phosphate; Pi, orthOphosphate; PPase, inorganic pyrophosphatase; aTP,
adenosine triphosphate; AMP, adenosine monophosphate; Tris, tris
(hydroxylmethyl) aminomethane; EDTA, ethylenediaminetetraaoetate.

20

21

within the last ten years a large variety of mixed acid adenylates, function-
ing as important metabolic intermediates, have been described (46). In the
formation of the mixed acid adenylates inorganic pyrophosphate is split

out from ATP and the reaction sequence is:

I. ATP + anion-‘—___—-.-_——_-7- acid adenylate + PP

Such a pyrophosphcmflytic reaction was demonstrated by Berg (47)
as occurring in the activation of acetate. In the presence of enzyme he
found that acetate was required for labeled PP exchange and that synthetic

adenyl acetate could be pyrophosphorylized to form ATP. Thus:

1rwmrw-‘W

 

II. ATP + acetate L— ,,adeny1 acetate + PP

Study of reaction II with 018 has established that the oxygen of the acetate
is transferred to the phosphate of the adenylate and has permitted the

formulation of the reaction as follows:

III.
0
5‘ . f 18
adenosine-O-P-O—P-O-P-O'=========aden031ne-O-g-O -g-CH3 + PP
0' 0‘
018
org-(5:0

in which the oxygen of the acetate makes an attack on the first phosphate
of ATP (48). If the same reaction mechanism is applied to the sulfate
activating-pyrophosphorylase reaction, then the formation of APS would

occur as shown in IV.

IV.
38% ° °
adenosine-O-.-O-.-O-.-O £=======;adenosine—O-P-O-$=O + PP
0">0' 0' 6' 0’
.75
O—§-O'

22
In this reaction the anionic attack is believed to involve an enzyme—
magnesium-sulfate complex (49).

(1.) Sulfuric-acid-adepyiate§3 A sulfuric adenylic acid anhydride
was first described in 1955 by H112 and Lipmann (50). The mixed anhydriie
was tentatively identified as APS and postulated to serve as the sulfate
donor (active sulfate) for the esterification of sulfate with phenol. In
subsequent work, however, Robbins and Lipmann (51) established that active

sulfate was not APS but adenosine-B'-phosphate—Siphosphosulfate, PAPS.

The metabolic relation between APS and PAPS was established by the elabora-

-’\j'i"hi ”II- .nwc—
‘ \ ‘q.

tion of the reaction sequence involved in their formation. Wilson and

Bandurski (52) and Robbins and Lipmann (53) showed that the reaction

v. ATP + 30"2 ’———:—7APS + PP

4

is followed by a second reaction
VI. APS + ATP := PAPS + ADP

which results in the synthesis of active sulfate, PAPS. The first reaction

is catalyzed by ATP sulfurylase and the second reaction by APS kinase.
During the course of separation of the ATP sulfurylase and APS

kinase enzyme, Robbins and Lipmann (44,53) obtained a third enzyme which

catalyzed the reaction

-2
4

VII. APS + Pi :F=========£=ADP + 80

The enzyme, ADP sulfurylase, was purified threefold from yeast extract and
freed of ATP sulfurylase. Activity determinations were made by incubation
with synthetically prepared APS (54) and measuring Pi disappearance, with
checks on ADP formation by electrophoresis or chromatography. While Robbins

and Lipmann have not investigated ADP sulfurylase extensively, they have

23

reported it to be unstable and to require no added cation activator.

(2,) gigsynthetic reactions involving éDP: At the present time
only two classes of enzyme catalyzed reactions are known in which a synthetic
or endergonic reaction is coupled to cleavage of phosphate from ADP (or
other dinucleotides). They are the synthesis of polyribonucleic acid and
the phosphate transferring reactions catalyzed by nucleoside monophosphate
kinases. The formation of APS from ADP may serve as a third example of
an ADP coupled reaction; however, as yet, this reaction has not been con—

clusively demonstrated. In RNA synthesis, the enzyme polynucleotide phos-

phorylase discovered by Ochoa (55) catalyzed the reaction:
VIII. nx—P-P —\.__—_———=‘ (x-P)n + nPi

in which x represents a nucleoside. In this reaction the cleavage of a
phosphate is followed by the formation of an ester bond.

The nucleoside monophosphate kinases can be considered to cata-
lyze the transfer of phosphate from a nucleoside triphosphate to a nucleo-
side monophosphate; or, in the reverse reaction, to catalyze the attack
of one nucleoside diphosphate on another to yield one molecule of nucleoside

monophosphate and one molecule of nucleoside triphosphate (56):

 

 

Nucleoside-A triphosphate + Nucleoside-B monophosphate
Nucleoside-A diphosphate + Nucleoside—B diphosphate.
The earliest known nucleoside monOphosphate kinase reaction is that cata-

lyzed by the ATP-AMP kinase, adenylate kinase (originally termed myokinase)(57):
IX. 2 ADP—=AMP + ATP

in which only adenine nucleoside phosphates are involved.
(3.) §ulfurylase_reactions“withgmolybdate; Work by Wilson and

Bandurski (45) has established that other group VI oxyanions, in addition

. I92? ’tJtJlj. In?“

(WW-.6:

 

24

to sulfate, can serve as substrates for ATP sulfurylase. The substitution

of M0022 for so;2 was found to lead to a much greater PP release from ATP,

and consequently could serve as a convenient means of assay for nTP sulfury—
lase.

Assuming that M0022 would also serve as a substrate for ADP
sulfurylase, the reaction

-2
M004

 

in which the anionic attack on ADP is made by molybdate, would be eXpected

. W‘s—5““ ‘Wm “—1

to occur. In this thesis, M0022 was used and P1 release measured to assay

“I- ‘

for ADP sulfurylase activity. g3

B . magpie. Pyrpphpspfiha t...a._s_..e.
Yeast pyrophosphorylase hydrolyzes the acid anhydride bond in
pyrophosphate and liberates two molecules of Pi for every molecule of pyro-

phosphate hydrolyzed:
XI, PP=2P1

The enzyme is specific for inorganic PP and will not hydrolyze other PP
linkages. Bailey and Webb (58) prepared highly purified yeast PPase

and found the presence of Mg+2 essential for activity, while Ca+2, Mn+2,
and Zn+2 were inhibitory in the presence of Mg+2. Divalent magnesium was
required as an activator in the yeast PPase preparations of Heppel and
Hilmoe (59). Kunitz (60) has crystallized yeast PPase and found in addi-
tion to Mg+2 activation a slight activation by Co+2 and Mn+2. In potatoes,
Nagawa, gt_§;, (61) report the occurrence of an acid and an alkali PPase
(pH optima of 5 and 8.5). The acid PPase is not activated by Mg+2 but not
inhibited by M0022. In either case no activation is obtained by Co+3,

Mn+2, or Zn+2,

25
2. MATERIAL AND METHODS
Pyrqphqsphqtase: Yeast PPase, prepared by the Heppel and Hilmoe
method, was supplied by Dr. Lloyd Wilson.

_§£eparation of adgnosine-phosphgsulfate; APS was synthesized

 

by the method of Baddiley, g§_§l.(54), 400 mg of dry pyridine - $03 complex
(prepared by Mr. Craig Squires) was slowly added with stirring to 200 mg
AMP and 350 mg NaHCO3 contained in 5 ml of water. The resulting reaction I
mixture was stirred for 10-20 minutes at 42°C.
The yield of APS was estimated by paper electrophoresis of a i

small quantity of the reaction mixture at 1-20 in M/lO pH 5.0 acetate

 

L] .

buffer. The nucleotide spots located by their uv absorption, were cut
out, eluted with water for 1/2 hour at 50-60°, and the concentration of
nucleotide determined from optical density readings at 260 m/J (62). Yields

of APS were obtained corresponding to about 8.5% of the AMP supplied.

.lsolation.of.AES. Previous attempts in this laboratory to isolate
aPS by means of the formate column described by Baddiley were unsuccessful.
The description by Brunngraber (63) of the isolation of PAPS by NaCl elution
from erbwex-l,chloride column, led to several unsuccessful attempts to iso-
late APS on ion exchange columns of this type. A new method for separa-
tion of nucleotides was suggested by the reported use of ion exchange resins
in determinations of stability constants of cation-nucleotide complexes
(64). Approximately 2 grams of 200—400 mesh, 10% crossed linked, chloride,
Dowex-l resin was formed into a 7.5 x 1 cm column. From 13 to lOO [/moles
of nucleotide in 20 ml of water were put on the column and eluted with a
solution containing .005 mole MnClz, .05 mole of NaCl, and .01 mole pH
8.2 tris—HCI per liter. The strength of the cation nucleotide complex de-

pends on the length of the phosphate chain in the nucleotide, and dinucleotides

 

 

?
i‘}

26

form Mn—complexes which are less strongly absorbed to the exchange resin
than those of mononucleotides. The Dowex column described was capable of
separating ADP, which was eluted first, from admixed aMP. However, it was
ineffective in separating APS from aMP because of the rapid hydrolysis of
APS, apparently catalyzed by Mn ion. Small quantities of APS could be
separated by use of a continuous flow electrophoresis apparatus built by
Mr. Robert Hamilton. Separation was accomplished by applying aPS reaction
mixture by means of a wick onto a hanging curtain of Whatman #3 paper.
M/201&15 acetate was employed as a solvent and a field strength of approxi-
mately 14.3 v/cm applied for 24 hours, while the apparatus was kept in a

10 cold room. The upper edges of the curtain were serrated to provide a

(ma; -‘vYJ'J mmcna-T

slower flow of solvent. The APS fraction was adsorbed on charcoal and
eluted according to the method given by Baddiley (54). alcohol was removed
from the eluate with a flash evaporator at 500 and the remaining solution
freeze-dried in a lypophil apparatus. a mixture of 4.2/uM APS and 4.6/uM

AMP was obtained.

Determination of OrthOphosphate. The method of Fiske and

-.0---‘---.--.C“----—.—.cfl.—.O---

Subbarrow (65) was used to determine inorganic phosphate.

EStiWQEiQQ-Q§;RCQEQi,' Samples were diluted with M/Z KCl and
shaken with an equal volume of 5% trichloroacetic acid. The resulting
turbidity was measured in a Klett—Summerson colorimeter with a #54 filter

and compared with crystallized bovine serum albumin as a standard.

Sulfurylase-assay. Sulfurylase activity was determined by

 

measuring molybdate stimulated release of P1. The reaction mixture con-
tained 50 ,umoles tris—HCl buffer, pH 7.5; 4IUmOles NaZMOOA, 0.3,umoles

EDTA; 2,0moles of either M3012 or other cation as specified, 2.0/omoles aDP

27

or aTP, and enzyme in a total volume of 0.50 ml. Parallel tubes were also

run without addition of molybdate. Incubation was for 60 minutes at 37°C.

Preparatigg‘g§_epgymg. Twelve grams of baker's yeast were sus-
pended in 100 m1 of .01 M pH 7.5 tris-HCl containing .001 M EDTA by grind—
ing in a porcelain mortar. This suspension was then stirred at 14,000 r.p.m.
in a high speed mixer for 10 minutes with 35 grams of 2001;) glass beads.
The extract was centrifuged, frozen, thawed, filtered through silk and
centrifuged again at 22,000 x g for 10 minutes. The supernatant liquid

was dialyzed for 20 hours at 1° against two one liter portions of .005 M

a: “turn! unnu- ‘1’"w-‘F-‘T
f

"a...

tris-HCl, .0001 M EDTA. A small precipitate was removed by centrifuging

5 Tana—‘7

l

for 10 minutes at 22,000 x g, and the supernatant liquid saved (stage II).

Heat_prgcipitgtigg. Dialyzed extract was heated to 49-500 while

 

being stirred, kept at this temperature for 2 minutes, then cooled in an
ice bath. The suspension was frozen, thawed, and centrifuged at 22,000

x g for 10 minutes (stage III). Loss of activity occurred quickly at
temperatures above 50°, and only 15% of the activity present after heating

at 49° was obtained following heating to 53°.

Acid_pgecipitation. Portions of the heated supernatant fraction

 

were dialyzed against acid buffer solutions. Dialysis was done against

250 volumes of 0.2 M acetate, 0.0005 M EDTA buffer of the appropriate pH

at 1°. After 3 hours the suspension was removed from the dialysis bag,

and centrifuged at 22,000 x g for 10 minutes. The precipitate was dissolved
in a volume of M/lO pH 7.5 tris—HCl equal to the supernatant volume. The
pH of the supernatant was recorded and found to range from 0.05 to 0.10

pH unit higher than the pH of the dialysis buffer employed.

28

Enzyme fractionatign. Table 5 summarizes the enzyme fraction—
ation. One unit of epzyve activity is defined as the amount of enzyme
which produced one /Jmole of inorganic phOsphats from nDP when incubated

-2
in the presence of M004 at 37° for one hour. All the fractions obtained
had greater activity with aTP than “DP. In addition pH precipitations

were made at several other pH values at which the concentration of protein

present was not measured. The activity of these fractions is given in Table 7.

“a
n

3. RESULTS

A. Release offiPhosphateCfrom aDP

-‘C- ‘--vm-.-

 

An enzymatic release of phosphate was observed when ADP was

DPS—IV.“ "Ira-'5' .M

V.

incubated with dialyzed yeast extract, Figure l. The increment in Pi
liberation obtained by addition of molybdate indicates the amount of sul-
furylase activity present (45).

Electrophoresis of the incubation mixture demonstrated the presence
of adenylate kinase in the enzyme preparation. Upon electrophoresis AMP,
ADP, and hTP Spots of approximately equal size were obtained. Furthermcre,
the incubation mixture was demonstrated to have PPase activity. Therefore,
the relative contributions of aTP sulfurylase and possible ADP sulfurylase
to the release of Pi could not be determined.

The effegt-of-cations. The release of Pi from ADP through the
action of aTP sulfurylase is dependent upon the presence of adenylate kinase

and PPase in addition to the sulfurylase enzyme. The following series of

reactions are involved:

XII. a. 2 aDP’—————-———'—-,ATP + AMP

-2
M004
b. ATP :;aMP + PP

 

C. PPE:========;2Pi

_.,_T

 

 

 

 

mm hoe omom «.0 0m unevenness» m.m ma .>H
as ems seem ~.ma on paasaaamaa. 00m .HHH
men saw Once m.0m Hm poappxo eeuaaaan .HH
00H w.am omen mm om pomppxo opsao .H
seepage
m wining Brian mmHosQ we as
epa>apoa seepage
anoboomm ofiuaomem mafia: Hence oesao> cowpompm seamen

 

ammo» Eoum hpfibfipod mmmahpsmasm mo cowpwowwwpsm

m mumda

 

Figure 1

Phosphate Liberation from ADP
As a Function of Enzyme Concentration

Lt? - 'xxzspm.‘ “I
'I
I
R

{staff's-:7; -
i .

d pmoles/Tube

 

 

 

00m

 

 

l

*4. ,Asli- » -

 

 

29
4

in which b.is probably a two step reaction involving an unstable AMP-M005
mixed acid anhydride. The requirement of Mg+2 by PPase for high activity
suggested elimination of magnesium ion from the incubation mixture might
serve to prevent liberation of Pi through the ATP sulfurylase route. The
-0
g

reaction APS + Pi-————>ADP + 804 is reported to proceed without the addi-

tion of any cation (44). In measuring the reverse reaction with molybdate:

 

-2
XIII. ADP + M004 .fisrAMP + Pi

with the dialyzed yeast extract, used in the study reported on in this
thesis, no appreciable release of Pi occurred in the absence of a cation.
The effect of no added cation and of several different divalent cations
on Pi liberation from ADP and ATP is shown in Table.6.

Substitution of Mn+2 for Mg+2 in the reaction mixture would be
expected to reveal the presence of ADP sulfurylase. Also, the removal of
any one of the enzymes shown in equations Xlla, XIIb, or XIIc from the enzyme
fraction being assayed would prevent Pi release by the ATP sulfurylase route
and permit the measurement of Pi liberated by ADP sulfurylase. Accordingly,
the enzyme preparation was fractionated at different pH values and assayed
in the presence of Mg+2 and Mn+2, ADP and ATP. All the fractions show very

2

similar ratios of ADP to ATP activity with either mh+ or Mg+2, Table 7.

I
--4,_. Jam“

8- Diva 1 eat 2 inc Aeiixetiee .af. .Rhes‘hate. 1:213:28; fro” 33011193121121:

 

Comparison of the PPase activity of stage IV enzyme and yeast
PPase prepared by the Hippel and Hilmoe method in the presence of different
activating cations disclosed an unexpected difference, Table 8. Both are
highly activated by Mg+2 and slightly activated by Mn+2 - in agreement with
the cation requirement of crystalline PPase (61). But PPase prepared by

the Hippel and Hilmoe method shows only a slight activation by zinc, whereas

_n - l2. '. .‘A 1' &-_ -!..a'4_~'_.‘n-u
_ .

i. __

 

Effect of Divalent Cations on Phosphate Liberation

 

 

 

Nucleotide and Cation Micromoles Phosphate 1

added Released i
ADP Mg+7~ 0.40
ADP Mn+2 0.21
ADP 2n+2 0.23
ADP None 0.05
ATP Mg+2 0.71
ATP Mn+2 0.51
ATP Zn+2 0.55
ATP None 0.07

 

Assay conditions as described for sulfurylase assay with nucleotides and

cations as indicated: 0.003 ml (9O/Vg) dialyzed enzyme.

 

‘ 'J‘“ ”lmfi
v

Ax"

'{m‘fTfiTfi/F

 

TABLE 7

Phosphate Release from ADP and ATP
with Mg+2 or Mn+2

 

Micromoles Phosphate per Tube

 

 

Enzyme 1
Fraction i
ADP . ATP

Mg/Mn Ratlo Mg/Mn Ratio i

‘ a.

Stage III i
50° supernatant 3.52/l.0 3.52 5.68/2.8 2.02

Stage IV

pH 5.5 supernatant 2.48/0.9a 2.53 5.84/3.02 1.93
pH 5.5 precipitate 0.86/0.20 4.30 1.02/O.64 1.59
pH 5.4 supernatant 0.92/0.36 2.56 1.20/0.76 1.58
pH 5.4 precipitate 2.40/0.4a 5.00 6.04/2.32 2.60
pH 5.2 precipitate 2.72/O.46 5.91 6.20/2.3 2.70

y”.-- -o

 

 

w v—-——

Conditions as described for sulfurylase assay, Mg+2 or Mn+2
.05 ml of enzyme fraction indicated.

, and

—1~:F
i

—-—
a

yr}... -‘1 u\..— :_V_.-

 

 

TABLE 8

Phosphate Liberation from Pyrophosphate
in the Presence of Divalent Cations

 

 

Enzyme
Cation
PPase Stage IV Enzyme Fraction
pmoles Pyroohosphate Liberated
Mg+2 2.02 2.04
hn+2 0.18 0.23
2n+2 0.30 1.92
None 0.06 0.07

 

 

 

Each tube contained inwpmoles: tris-HCI, pH 7.5, 50; NagMoO , 5;
EDTA, 0.3; cation, 2.0; and either 651pg of stage IV enzyme raction
or PPase in a total volume of 0.5 m1. Incubation was at 37°C for

60 minutes.

,rtgm. Oll‘: Pm‘ww
l i’
2 . ;

30

stage IV enzyme exhibits a high Zn+2 activation. The stage IV enzyme shows
no phosphatase activity on ADP or nTP (in the absence of M0022). Thus
the presence of a yeast PPase is suggested, which may be differentiated
from the previously reported pyronhosphatases on the basis of its activa—
tion by divalent zinc.
4. DISCUSSION

The reaction APS + Pi-———a>ADP + 3022 catalyzed by ADP sulfury-
lase is known to occur (44), although it has been only investigated briefly.

The writer wished to study the ADP sulfurylase reaction in the opposite

-2
direction, ADP + SOA-————4>»APS + Pi , by use of the molybdate assay (45).

lrl‘rfijrvm 5.3-? I!

Since M0022 has been shown to SUPStitute for 8022 in the reaction catalyzed
by ATP sulfurylase, the occurrence of the reaction ADP-—————;-4>M IIP + Pi
would be considered to demonstrate the presence of ADP sulfurylase. A
M0022 dependent splitting of Pi from aDP was measured; however, a larger
amount of Pi was released from aTP than from ADP with every enzyme fraction
assayed.

-2
ADP incubated with M004 and yeast extract may undergo the

reactions shown in Figure 2.

----—-——.—.---

Figure 2. Reactions of ADP, aTP, and Molybdate

ADP AMP + PP

1' \M -/ 3.1
M00.

2/
AMP+ATP AMP 2F:

 

Reaction (1) is catalyzed by adenylate kinase. (2 ) by“ T? sulfurylase,
(3) by PPase, and (A) is that postulated for “DP sulfurylase.

31

Starting with ADP it is evident that Pi may be released by ADP sulfurylase;
or phosphate may be released by means of an ATP sulfurylase route.
The ratio of molybdate stimulated phosphate release from ATP and ADP was
generally about 2:1 in all enzyme preparations. Fractionation of the enzyme
preparation by dialysis against buffers of different pH values, in an at~
tempt to separate ATP sulfurylase, PPase, or adenylate kinase from pcssitle
ADP sulfurylase, failed to change appreciably the ratio of Pi released from
the two nucleotides. Substitution of Mn+2, an ion which is a poor activator
of PPase, for Mg+2 was regularly employed in an attempt to demonstrate ADP
sulfurylase activity, but manganese decreased approximately proportionately
the release of Pi from both ADP and ATP.

Thus, in yeast extracts, M0022 does not bring about the reaction
ADP + M05E3—————> AMP + Pi indicating either the failure of M0022 to
substitute for $022 or the irreversibility of ADP sulfurylase. While in
theory all reactions are reversible, many in fact are effectively irrevers-
ible because they do not proceed to any appreciable extent in one direction.
The ADP sulfurylase reaction has previously been measured only in the direc-
tion of the phosphorclytic attack on APS (44,53,66). The reverse reaction,

applying the reaction mechanism which has been postulated for the formation

of nucleoside acid anhydrides, is depicted as:

XIV;
9
adenosine -0-P—O-P—0-————————e> adenosine—O-P-O-S=O + Pi
0‘ 0' 0‘ 0'
0
'0-$=0
0'

and this reaction would be unique in that no other enzyme catalyzed reac-
tion has the formation of a mixed mononucleotide acid anhydride been deman-
strated to be coupled with the cleavage of a high energy phosphate of a

nucleoside diphosphate,

32
The writer was not successful in separating APS from ADP by
means of ion exchange columns; but with Mn+2 used as an eluant ADP can
be displaced from a cation exchange column ahead of admixed AMP. Under
the same conditions a hydrolysis of APS occurs, probably catalyzed by the
manganese ion. Continuous flow electrophoresis, while convenient, can be

used only for separating small quantities of AMP and APS.

 

 

Yeast inorganic pyronhosphatase is reported to be chiefly acti- ?*“
i
vated by Mg+2, with slight activation by Mn+2 and Co"2 (60). A pyro- .
phorvntamaactivated by zinc ion has not been reported. The Zn+2 activated L
release of Pi from inorganic pyrophosphate, which was reported on in this '
thesis, with pH 5.5 supernatant enzyme is therefore of interest. This yeast El}

enzyme fraction catalyzes only small amounts of Pi release from PP in the
presence of Mn+2, but catalyzes much greater amounts in the presence of
Mg+2 or Zn+2. Although a zinc activated PPase is thereby indicated, the
extract has not been fractionated further with the purpose of separating
Zn+2 dependent activity from Mg+2 dependent activity.

Very recentlychhlesinger and Coon (67) also reported a yeast
pyrophosphatase preparation which catalyzed the release of Pi from PP with
either Zn ion or Mg ion as the activator. Furthermore, they found that

the same preparation possessed nucleotidase activity; however, nucleotides

were hydrolyzed at a very much slower rate then was inorganic pyrophosphate.

 

SUMMARY

1. The deoxyribonucleic acid (DNn) mediated genetic transforma-
tion of bacteria was discovered over 15 years ago. There have also been
some reports of the apparent genetic alteration of bacteria and yeastsby
ribonucleic acid. Transformations by DNA have not been demonstrated to
occur in other organisms with the exception of the recent report of
Oppenoarth's on yeast transformation (43). In the writer's experiments
the genetic transformation of yeastswas attempted utilizing adenine mutants

of Saccharomyces. The results of a single experiment are reported in which

 

adenine requiring cells were treated with a crude extract of adenine inde-
pendent cells. The treated cells, shaken for 9 hours with the crude ex-
tract, showed a greater number of cell reversions to adenine independence

than those not so treated. These preliminary results suggest that for further
experiments on yeast transformation, the mutants at the adenine loci in

Saccharomyces may prove useful. The adenine dependent, red pigmented mu-

 

tants may be treated with extracts from the white, adenine requiring proto-
trophs, plated on adenineless medium, and the colonies appearing, which
represent reversions to adenine independence, counted after only a few days
incubation. As a further check, the colonies which represent reversions
or transformations must be white and not red.

A procedure for extraction of nucleic acids from yeastsis described
in which an acetone powder in a sodium chloride - sodium dodecrl sulfate
solution is stirred at a high speed with glass beads. Following removal
of protein, the extract can be enriched in deoxyribonucleic acid by pre-

ciritating ribonucleic acid with a calcium chloride solution.

II. adenosine diphosphate sulfurylase has been reported to occur
in yeast and to catalyze the phosphorolysis of adenosine phosphosulfate (“PS)

33

34

to adenosine diphosphate (“DP) and inorganic sulfate.
_ . -2

a.) .1138 + Pi ——-—-)nDP + 304
The present eXperiments were designed to test for the occurrence of the
back reaction by making use of the ability of molybdate to serve as an
analog of sulfate. Since the resultant phosphato-molybdate compound might
be expected to be unstable the reaction expected was:
2

b.) aDP + {.1002 —————> AMP + Pi + P110022 37”“?

Although a molybdate dependent enzymatic cleavage of Pi from aDP was ob-

tained, a still larger release of Pi occurred whenever the enzyme fractions

were incubated with ATP. In the presence of adenylate kinase, pyrophos-

ll. _'. A' 2'": il- -"',_‘:vfj.‘~ ‘2- "

phatase, and ATP sulfurylase, phosphate can be released from ADP. It was

not possible to eliminate this means of Pi release from aDP by the substi-
tution of manganese ion for magnesium ion nor could any of the three enzymes
be removed from the enzyme preparation. Thus the ability of ADP sulfurylase,
from baker's yeast, to catalyze reaction b could not be established, indi-
cating either the failure of $0022 to substitute for sulfate or the irre-
versibility of ADP sulfurylase.

During the course of the experiments a divalent zinc ion activated
enzymatic release of phosphate from inorganic perphosphate was observed.
The presence of a yeast pyrophosphatase is therefore suggested which may
be differentiated from previously reported pyronhosphatases on the basis

of its activation by divalent zinc.

 

3.

4.

10.

11.

14.

15.

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Natl. Acad. Sc.

.II..--‘-.-.

(11.5.) Ag: 1053-1057.

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Biochim. et Bioohys. Acta. 41: 30—36.

 

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