MECHANISMS OF PTERIDINE-OMMOCHROME INTERACTIONS
IN DROSOPHILA MELANOCASTER
By
Joseph B . Cirone Jr.
A THESIS
Submitted to the Faculty of the Graduate School of
the Creighton University in Partial Fulfillment
of the Requirements for the Degree of Master
of Science in the Department of Biology
Omaha, 1974
Thesis Approved
3y
Major Advisor
Dean
4ÚJMNI aüEMOmi LIBRARYCreighton University
Omaha, Nebraska 6817S378400
V
FORWARD
I am sincerely grateful to my major thesis advisor,
Dr. Harry Nickla, for his never ending assistance in the
preparation of this thesis and consultation during the
course of investigation. I would like to thank
Dr. Charles B. Curtin and Dr. Robert W. Belknap for
their aid in preparing the manuscrpt. Thanks are due to
my family for their love and inspiration. I wish to
extend my appreciation to my friends, Mary Zimmer,
Larry Pribyl and Dick Talboy for their encouragement
and interest.
TABLE OF CONTENTS
Page
FORWARD v
I. INTRODUCTION 1
II. REVIEW OF LITERATURE 3
III. MATERIALS AND METHODS 13
IV. RESULTS AND DISCUSSION 20
V. SUMMARY 40
VI. LITERATURE-CITED 42
VII
Figure
LIST OF FIGURES
Page
1. Pteridine pathway proposed byKaufman (1967a). 6
Figure 2. Pteridine pathway proposed byFukishima (1970). 7
Figure 3. Pteridine pathway modifiedfrom Rembold (1970). 8
Figure 4. Results obtained from eyes of female wild-type and eye- color mutants of Drosophila melanogaster. 23
Figure 5. Results obtained from testes and Malpighian tubes of male wild-type and eye-color mutants of Drosophila melanogaster. 25
F igur e 6. Points of interaction between the ommochrome and pteridine pathways according to the mechanisms proposed. 36
viii
LIST OF TABLES
PageTable 1. Number of replications
performed for eyes, testes and Malpighian tubes, for fourteen strains of Drosophila melanogaster. 17
INTRODUCTIONI .Ommochromes (brown pigments) and certain
pteridines (red pigments), contribute to the typical
reddish-brown eye color of wild-type Drosophila
melanogas ter. Each pigment is the end product of a
seemingly unrelated metabolic pathway (Ziegler 1961;
Kaufman 1967a). Mutations which alter the synthesis
of these pigments are manifested as numerous
variations in eye color (Linds ley and Grell 1967).
Suppressed production of brown pigment results in
the accumulation of ommochrome precursors which in
some instances alters the production of drosopterins
and their intermediates. "Pteridine mutants"
accumulate pteridine intermediates and show altered
ommochrome formation (Hadorn and Mitchell 1951) .
Therefore, an interaction between the two biosynthetic
pathways is indicated.
This investigation is a preliminary attempt to
determine the nature of the interaction between the
ommochrome and pteridine metabolic pathways. To
elucidate the basis of this interaction , tryptophan,
N —formylkynurenine, kynurenine, 3-hydroxykynurenine,
phenylalanine, and tyrosine, were separately fed to
larvae of wild-type and eye-color mutants of D.
2
melanogaster. Changes in ptaridine accumulation
resulting from these feedings were determined in eyes
and testes using chromatographic and fluorometrie
methods (Hadorn and Mitchell 1951). Since riboflavin
accumulation is influenced by alterations in eye
pigment synthesis (Nickla 1972; 1973), levels of this
compound were also determined in the appropriate
tissue, the Malpighian tubes.
II. REVIEW OF LITERATURE
Studies which examined the synthesis and
deposition of the ommochrome and pteridine pigments
in ]}. melanogas ter have provided considerable in
formation on gene action (Ziegler 1961; Kaufman
1967a). The two pigments can be distinguished on
the basis of their solubility in various solvents and
the time at which they appear during development.
P te ri dines, which accumulate approximately 71 hours
after pupation (Hadorn and Ziegler 1958), are water
soluble and can be ex t rae ted fro m eyes wit h ethanol
(Mainx 1938; Ephrussi and HeroId 1944). 0mmochromes
do not dissolve in wa ter (Mainx 19 38) and can be
extract ed with methan ol and HC1 (Ephruss i and Herold
1944). They appear in the eyes approximat ely 54
hours after pup arium formation (Danneel 19 41) .
P i gment continues to accumulate. •for 2 to 3 day s after
eclosio n (Chauhan and Robertson 1966) .
A third pigment of interest in this s tudy is
riboflavin, which acc umulates in the Malpi ghian tubes
(Nickla 1972) and is a dietary requirement of D.
melanogaster (Sang 19 56 ) . Rib of lavin is n orman yp resent in the tube cells between the sixt eenth and
eightee nth hour of de velopment (Demerec 19 65) . The
4
relationship between riboflavin and the biosynthesis
of pteridines will be discussed subsequently.
PTERIDINES
In addition to the eyes, pteridines accumulate
in the testes sheath, fat bodies and Malpighian tubes
of wild-type J). melano gas ter. Isoxanthopterin is
detectable in all three of these tissues in second-
instar larvae. However, small amounts of other
pteridines may also be found. The highest concen
tration of isoxanthopterin is in the Malpighian tubes
(Hadorn and Mitchell 19 51) . During pupation, large
quantities of pteridines accumulate just prior to
final pigment synthesis. It is during this period
that coloration of testes occurs (Stern 1941a, b ).
There are two classes of pteridines, conjugated
and unconjugated. Conjugated pteridines belong to
the folic acid derivatives and are found with p-amino-
benzoylglutamic acid attached to the pteridine ring.
All other pteridines are termed unconjugated (Kaufman
1967a). The pteridine ring system appears below
(from Kaufman 1967a): 4 5
Many unconjugated pteridines are required cofactors
for a number of hydroxylation reactions (Kaufman
1967a, b ) .
5
Due to a variety of presumptive roles, bio
synthesis of pteridines has been the subject of ex
tensive research. Albert (1957) demonstrated the
feasibility of the conversion of purines to pteridines.
The primary step in the transformation involves release
of a formate molecule from a guanine nucleotide.
Xanthopterin and neopterin are among products of the
reaction (Guroff and Strenkoski 1966).
Experiments indicate close metabolic relation
ships among pteridines which accumulate in the various
tissues of 13. melanogas ter . Eye-color mutants not
only exhibit altered pteridine accumulation in eyes,
but also in other tissues which possess these com
pounds (Hadorn and Mitchell 1951). A pathway based
upon data accumulated through the study of mutant D.
melanogas ter has been proposed for pte ridines by
Kaufman (1967a) and appears in Figure 1. Recently,
Fukushima (1970) and Rembold (1970) (Figures 2 and 3),
proposed biosynthetic schemes which resolve certain
inconsistencies in the pathway proposed by Kaufman
(1967a). If these two schemes are linked at the
6
GUANINE NUCLEOTIDE
6
NEOPTERIN
-HYDROXY-METHYLPTERIN FOLIO ACID
2-AMINO-4-HYDROXYPTERIDINE---------------- - SEPI APTE RIN
ISOXANTHOPTERIN BIOPTERIN
DROSOPTERIN
Figure 1. Pteridine pathway proposed by Kaufman (19 6 7a) .
7
GUANOSINE TRIPHOSPHATE
7,8-DIHYDRONEOPTERIN TRIPHOSPHATE
L-THREO-DIHYDRONEOPTERIN
L-THREO-DIHYDROB10PTERIN
SEPTAPTERIN
DIHYDROBIOPTE RIN
BIOPTERIN
Figure 2. P t eridine pathway proposed by Fukushima (1970).
8
7,8-DIHYDRONEOPTERIN
QUIONO ID DIHYDRON SOFTER IN-
7,8-DIHYDROPTERIN
6-HYDROXY-7,8-DIHYDROPTERIN
DIHYDROXYXANTHOPTE RIN
XANTHOPTERIN
LEUCOPTERIN
PTERIN
ISOXANTHOPTERIN
+NEOPTE RIN
Figure 3. P teridine pathway modified from Remb old (19 70).
9
guanine nucleotide, then variable accumulations or
deficiencies in substances such as isoxanthopterin
without expected changes in sepiapterin and biopterin
may be explained.
Several enzymes which catalyze steps in the syn
thesis of pteridines have been studied. Xanthine
dehydrogenase (XDH), required for the conversion of
2-amino-4-hydroxypteridine (pterin) to isoxanthopterin,
results from a complementation of allelic products.
Classman e£ aj . (1965) found that flies which are
homozygous maroon-like or maroon-like-bronzy have
mutant eye color and lack XDH activity. However,
females heterozygous for these two alleles (maroon-
like /maroon-like -bronzy) have wild-type eye color
and 10% wild-type XDH activity. Studies of Horikawa
at al. (1967) indicate that uric acid, a product of
XDH action, is a non-competitive inhibitor of XDH.
Nutritional controls have been recognized for XDH.
Collins e t al. (19 70) found an increase in XDH
activity when flies were reared on synthetic semi-
defined media which had been supplemented with amino
acids. Electrophoretic species of XDH have been
described (Yen and Glas s man 196 7) and riboflavin is
a required cofactor for its activity (Chovnick and
10
S ang 19 6 8) .
Sepiapterin reductase , which is involved in the
conversion of sepiapterin to biopterin has been
examined in rat liver preparations (Matsubara et al.
1966) and in I). melanogas ter (Taira 1961). In
addition, the reduction of dihydrobiopterin to tetra-
hydrobiopterin, an important cofactor in several
hydroxylation reactions, is catalyzed by dihydro-
pteridine reductase (Kaufman 1959; 1967b). The final
step in the synthesis of drosopterins takes place on
pigment granules found in the eyes. The granules are
composed of a ribonucleic acid-protein complex
(Ziegler 1961).
Pteridines have been metabolically related to
riboflavin in grasshoppers (Bodine and Fitzgerald
1948) and yeast (McNutt and Forrest 1958). Although
D_. melanogas ter can synthesize pteridines, riboflavin
is a dietary requirement (Sang 1956). Since eye-
color mutants often exhibit variation in Malpighian
tube color (Brehme 1941), Nickla (1972) suggested
that a common mechanism may influence pteridine syn
thesis and riboflavin accumulation in the Malpighian
tubes. The mechanism of interaction is yet to be
determined.
11
OMMOCHROMES
From studies of a number of researchers (Beadle
and Ephrussi 19 35 ; Beadle 19 37a, b; Tatum and Haagen-
Smit 1941; Butenandt et_ al. 1949; Green 1949; Danneel
and Zimmermann 1954), Ziegler (1961) has proposed the
following biosynthetic pathway for the production of
ommochromes.
I II IIITRYPTOPHAN--♦ N-FORMYLKYNURENINE---- - KYNURENINE---- -
IV3-HYDROXYKYNURENINE---♦ OMMOCHROME
Butenandt and Nuebert (1958) established the
structures of ommochromes. Two forms were reported
to exist-, alkali sensitive ommatins and. alkali stable
ommins. A particular ommatin, dihydroxanthommatin,
constitutes the major portion of the brown pigment
in the eyes of wild- type I). me 1 an o gas ter (Phillips
et al. 1973) .
Enzymes Tfhich catalyze steps in the ommo chrome
pathway are, tryptophan pyrrolase (Step I) (Knox and
Mehler 1950; Baglioni 1959), kynurenine formidase
(Step II) (Classman 19 56) , kynurenine-3-hydroxylase
(Step III) (Ghosh and Forrest 19 6 7) and phenoxaz inone
synthetase (Step IV) (Phillips and Forrest 1970).
12
Two of these enzymes have been subjected to extensive
research. Tryptophan pyrrolase, the structural
product of the vermilion'7' gene (Baillie and Chovnick
1971), is regulated by an inducible system and is
found at its highest activity in the anterior portion
of the fat body (Rizki and Rizki 1963). Kynurenine-3-
hydroxylase is located in the outer membrane of
mitochondria in Meurospora cras s a (Cassidy and Wagner
1971) and D. malano gas ter (Ghosh and Forrest 19 6 7) ,
where its highest activity is in the Malpighian tubes
just prior to pupation (Hend ri chs -He rt el e_t_ al_. 1969).
Granules on which final synthesis of ommochromes
occurs are similar in structure to the granules which
carry drosop t erins. However, the two types of
granules are cytologically distinct (Shoup 19 6 5) .
Phillips et al. (19 73) suggested phenoxazinone syn
thetase is associated with particles which resemble
the pigment granules.
III. MATERIALS AND METHODS
Strains of _D. melanogaster used in this investi
gation were wild-type (Urb ana) , clot (cl) , maroon-like
(ma-1) , garnet ( ) , mahogany (mah) , raspberry (ras") ,
light (It), vermilion (v) , cinnabar (cn) , scarlet (s t ) ,
carnation (car) , sepia (se), carmine (cm) , and p rune
(pn) . Each strain was maintained at 2 4±1° C in half
pint milk bottles containing standard a gar- c o m m e al
fa rewer's yeast-molasses-sucrose-propionic acid medium.
To minimize any influence age and crowding may have on
pteridine accumulation (Chauhan and Robertson 1966) ,
the following preparation and collection procedures
were employed. Adu j-t flies were, allowed to lay eggs on
lids of S tender dishes containing approximately 5cc of
standard medium which was covered with a circular piece
of paper toweling. Forty eggs from each strain were
collected from the Stender dishes and placed into a
50mm x 5mm vial which contained 0.3cc of standard
medium. Twenty-four vials were prepared for each
strain. Of the twenty-four vials, three were controls
and three were non-treated. At appropriate times 50
microliter aliquots of water were added to the control
vials. Nothing was added to n on-1 re a ted vials. The
remaining eighteen vials, divided into six groups of
14
three each, were "treatment" vials. Groups 1 through 6
were supplemented with tryptophan, N-formylkynurenine,
kynurenine, 3-hydroxykynurenine, tyrosine, and phenyl
alanine , respectively. Five of the compounds were
administered at 10 M concentrations in 50 microliter
aliquots. N-formylkynurenine did not completely dis
solve in water and was therefore administered as a 50
microliter suspension. Through preliminary experiments
it was found that larvae would die if the treatments
were administered prior to 33+2 hours after hatching.
Therefore, two feedings were administered, one at
33±2 hours and the other at 74+2 hours after hatching.
When pupae had formed, the three 50mm x 5mm vials of
each group were placed into one 2.5cm x 10cm vial
which contained 10 cc of standard medium. Thus,
sufficient space was provided in the large vial so
that as flies emerged from puparia they had enough
room to survive. When newly emerged flies were two
days old, they were etherized and transferred to fresh
2.5cm x 10cm vials. Flies were collected for chromato
graphic analysis when they were 6 ±1 days old.
Green (1949; 1952) performed experiments which
showed that vermilion flies, which normally do not pro
duce ommochromes, are able to form these substances if
15
fed N-formylkynurenine, kynurenine or 3-hydroxy-
kynurenine. In this investigation preliminary ex
periments were performed in which hrown-vermilion flies
which have white eyes, were fed N-formylkynurenine,
kynurenine, and 3-hydroxykynurenine respectively. The
resulting flies had brown eyes. This indicated that
the treatments were effective at the concentrations
administered. However, it was noted that the eyes of
the brown-vermilion flies which were fed N-formyl-
kynurenine were not as dark brown as the eyes of the
fli.es which were fed kynurenine or 3-hydroxykynurenine.
This was attributed to the fact that the N-formyl-
kynurenine did not completely dissolve in water.
Therefore, larvae may not have consumed as much
N-formylkynurenine as compared to amounts of kynurenine
or 3-hydroxykynurenine consumed.
Chromât o graph y.
The chromatographic procedures followed those of
Hadorn and Mitchell (1951) with some modifications.
Eyes from female flies and Malpighian tubes and testes
from male flies were used for chromatography. Separate
chromatograms were made for each type of tissue
examined. Whatman //I chromatography paper was prepared
by drawing a pencil line 1% . inches from the bottom of
16
each sheet. Spaces were reserved on the line for six
treatments, a non-fed treatment, a control and a blank.
For each chromatogram, tissue to be examined was
removed with a forceps and placed randomly (coded by
random numbers) at 22mm intervals along the line. The
tissue was then squashed onto the paper with a clean
glass rod.
The chromatographic procedures outlined were
performed in ten replications for each tissue studied.
However, due to low viability in some flies, and the
loss of single measurements in others, fewer repli
cations were performed and missing measurements were
estimated in some instances., This information is
contained in Table 1.
When squashing procedures were complete, chromato
grams were rolled into cylinders and stapled along the
edges approximately three inches from the top and
bottom. Cylinders were placed into Gin x 1 Sin chroma
tography jars containing 150ml of developing solution
(N-propanol and 5% ammonia, 2:1). Jars had been
equilibrated for at least one hour before chroma
tography was initiated. After 5 to 6 hours the
chromatograms were removed, air-dried and developed
a second time in the same developing solution. All
17
EYES TESTESMALPIGHIAN
TUBEScl 10 10 10ma-1 9 9 PHE 0
& 2 10 10 0mah 10 C 10 0
2r as 10 NFK 10 10It 10 10 0V 10 10 0cn 10 10 0s_t 8 10 10car 10 10 10se 10 TYR 10 10cm 8 8 0
2R 10 10 0wild 8 9 9
Table 1: Number of replications performed for eyes,testes , and Malpi ghian tubes for 14 strainsof D . melanogas ter. A treatment name ;
control (C), N-formylkynurenine (NFK),
tyrosine (TYR), phenylalanine (PHE),
appearing after the replication number
indicates a missing measurement for that
compound.
18
developing procedures were performed in the dark.
Pteridines were located on the chromatograms with
an ultraviolet lamp and fluorescent colors (Gregg and
Smucker 1965; Kadorn and Mitchell 1951). The Rf values
calculated for the pteridines examined are as follows:
drosopterin (0.05), isoxanthopterin (0.22), xanthopterin
(0.30), sepiapterin and riboflavin (0.50), and pterin
and biopterin (0.58). To prepare for fluorometry, the
chromatograms were cut horizontally between each row of
pteridines.
Fluorometry
A Turner Model 111 fluorometer, fitted with a door
for reading paper chromatograms, was used to quantify
pteridine spots. The primary filter used for all
readings was number 7-60 (filter numbers are those
supplied by Turner). Combinations of secondary filters,
used are as follows: Drosopterins, 2A and 23A;
xanthopterin and isoxanthopterin, 2A, 58 and 1-60;
isosepiapterin, sepiapterin and riboflavin, 2A and 58;
pterin and biopterin 2A and 75 (Wright and Handly
1966) .
Statistical Me thods
Experimental groups were arranged according to the
randomized-b1ock design. Analysis of variance was
19
employed in the interpretation of results. Missing
measurements were estimated according to the procedures
outlined by Woolf (1968).
IV. RESULTS AND DISCUSSION
In this investigation the eyes , testes, and
Malpighian tubes of fourteen strains (eye-color mutants
and wild-type) of D . melanogas ter were examined for
interactions between the ommochrome and p t e ri din e
metabolic pathways. In addition, possible inter
relationships between pteridine synthesis and rib o-
avin accumulation in the Malpighian tubes were studied.
The experimental procedure included the separate
feeding of tryptophan, N-f ormyIkynurenine , kynurenine,
3-hydroxykynurenine, tyrosine, and phenylalanine to the
experimental organisms. Non-treated flies and controls
were also reared. Chromatographic and fluorometric
procedures employed were designed to obviate the
effects oi each treatment on pteridine accumulations as
compared to amounts found in controls. To reduce
variation in pteridine accumulation due to effects
other than the treatments administered, flies were
reared at a constant temperature and with a uniform
degree of crowding.
figure 4 illustrates the changes observed In
pteridine accumulations as compared to control values
in the eyes of females from all strains of D.
21
melanogas ter examined. Figure 5 presents the results
obtained from the testes and Malpighian tubes of male
flies. In the presentation of results which follows,
trends in pteridine accumulation which are significant
at the .05 probability level will be emphasized. If
no trends exist the results will be referred to as
variable. . .
Mutants examined in this investigation can be
divided into four categories according to the amounts
of ommochromes and drosopterins accumulated in adults.
(1) Increased brown; reduced red, (2) reduced brown;
normal red, (3) normal brown; reduced red, and (4)
reduced brown and reduced red (Linds ley and Grell 1967;
Brown 1973).
Group 1 from above contained mah, ras2 , pn , se
and cm mutants. In general, when these flies were fed
the ommochrome precursors, their eyes exhibited
variable alterations in pteridine content. However,2 demonstrated significant increases in drosopterins.
Testes of group 1 mutants also exhibited variable
results. Riboflavin content was examined in Malphighian
tubes of ras and se flies . In both cases riboflavin
content rose when the flies were treated with N-formyl-
kynurenine. However, no overall trends were observed
Figure 4. Amounts of drosopterin, isoxanthopterin
and xanthopterin, sepiapterin, biopterin
and pterin (HB), and isosepiapterin in the
eyes of female c 1, ma-1, } mah , ras ,
It , cn > s t, c ar , s e , cm, pn and wild-
type IK me1anogas ter. Treatments appear
in the following order: tryptophan, N-.
formylkynurenine, kynurenine, 3-hydroxy-
kynurenine, tyrosine, phenylalanine and
non-treated. Numbers after each group
are standard errors. The denotes
significance at the .05 probability level.
Figure 5. Amounts of isoxanthopterin, xanthopterin,
sepiapterin and biopterin and pterin (HB)
in the testes and riboflavin in the
Malpighian tubes as compared to controlOlevels found in male cl, ma-1, , mah,
ras2 , It, v, cn, st, car, se, cm, pn and
wild-type _D. melan'ogas ter. Treatments
appear in the following order: tryptophan,
N-formylkynurenine, kynurenine, 3-hydroxy-
kynurenine, tyrosine, phenylalanine and
non-treated. The numbers after each group
are the standard errors. The denotes
significance at the .05 probability level.
26
for riboflavin accumulations in flies fed the ommo-
chronie precursors . Variable patterns in pteridine
accumulations were observed in group 1 flies when fed
phenylalanine or tyrosine. This was also the case for
non-treated group 1 flies.
Vermilion, cn and st were the mutants examined
from group 2. The results obtained from the eyes of
this group were variable. However, cn mutants demon
strated significant increases of sepiapterin when fed
3-hydroxykynurenine and increased HB when tryptophan
was administered. The tastes of group 2 flies did not
show any trends in pteridine alteration. No changes
were seen in the riboflavin accumulations in the
Malpighian tubes of s_t mutants . Group 2 flies ex
hibited variable trends in pteridine accumulations
when fed tyrosine and phenylalanine. No alterations in
pteridine levels were observed for non-treated group 2
mutants.
Clot flies were the only members of group 3. All
three tissues examined in this mutant showed variable
results for all treatments.
Flies which belonged to group 4 were ma-1, _g_ , It
and car. Eyes of three of these mutants , ma-1, an<j
lt_, showed significantly increased accumulations of
pteridines when fed the ommochrome precursors. The
results obtained from car flies were variable. The
testes of the group 4 flies, especially ma-1, demon
strated non-significant decreases in pteridine content.
However, with tryptophan as the treatment no alteration
in pteridine accumulation was observed in ma-1, It and 2
S flies . No trends in riboflavin accumulation were
seen in the Malpighian tubes of car flies . The testes
of group 4 flies showed reduced pteridines when fed
phenylalanine and tyrosine. These treatments caused
no significant changes in eye compounds for group 4
mutants. Non-treated group 4 flies did not show
alterations in pteridine quantities.
Wild-type flies did not show trends in any of the
tissues examined for any treatment.
Three assumptions were made at the outset of this
investigation. The first assumption was that there is
an interaction oetween the ommo c h come and pteridine
metabolic pathways in D. melanogas ter. Support for
this assumption has been presented in the review of
literature. That a build-up of ommo chrome precursors
could be accomplished in I). me lanogas ter by feeding
precursors to larvae was the. second assumption.
Support for this assumption had been previously
27
28
presented (Green 1949 ; 19 52) , and subsequent support
was gained through preliminary experiments (see:
Materials and Methods). The third assumption was that
the build-up of ommochrome precursors could alter
pteridine accumulation in I), melanogas ter. The results
obtained from the eyes of ma-1, ¿2-, lt_ and ras2 mutants
support this third assumption.
Results of Brown (1973) were used to determine the
biochemical similarities among ma-1, g2 , lt_ and ras2
flies. Brown’s methods compared both pteridine inter
mediate and ommochrome precursor levels of the same
strains of flies used in this investigation to levels
found in wild-type I). melanogas ter. Brown examined
third-instar larvae, pupae, and adults. Male and
female flies were studied separately. Pupae were
examined at 48 hours after pupation, at which time
there is normally a large accumulation of precursors
(Ziegler 1961). Brown found that during this period
pupae of ma-1, It and _g2 females did not exhibit an
accumulation of precursors. All of the other flies
examined showed amounts of ommochrome precursors and
pteridine intermediates that approached or exceeded
levels found in wild-type. In some cases, as in cm
females, the quantities of ommochrome precursors were
29
low, but the amounts of p teridines were close to
wild-type. Quantities observed in ma-1, It and j*2
remained reduced through the adult stage. In addition,
drosopterin and ommochrome levels in the eyes of ma-1,2It and g_ females were well below the quantities
observed in wild- type EK melanogas ter.
From similarities found in female ma-1 , It and ¿2-
mutants, the following conditions will be considered as
requisite for a fly to elicite a perceivable alteration
in pteridine accumulation in response to feeding ommo
chrome precursors. (1) During puparium formation the
accumulation of both ommochrome precursors and
pteridine intermediates must be substantially reduced
from wild-type levels. (2) In adult flies the quan
tities of ommochrome precursors and pteridine inter
mediates must remain below amounts observed in wild-
type flies. (3) Both drosopterin and ommochrome levels
in the eyes of adults must be reduced as compared to
levels in wild-type flies. Female ma-1 , It and g2
mutants were the only flies examined which met all of
the above conditions. The fact that no male flies meet
these conditions could explain why the effects of the
treatments were not perceivable in the testes and
Malpighian tubes. Since the levels of ommochrome
30
precursors were low in ma-1, It and j*2 mutants, feeding
ommochroma precursors could increase quantities of
these compounds in flies, thereby enhancing detection
of the interaction between the ommochrome and p teridine
pathways. All other mutants examined normally have
high ommochrome precursor levels, therefore, treat
ments would not be capable of increasing quantities of
ommochrome precursors in these flies . Experimentally
increasing levels of ommochrome precursors increased
pteridine accumulation. Ziegler (1961) pointed out
that D. me 1anogas ter tend to lose pteridines after
maximum levels have been reached a few days after
eclosion. Therefore, the t re atment-in du ce d increase
in pteridines would eventually be lost in adult flies .
If the pteridine content was high in adults, the
treatment-induced increase could be lost before
chromatography could be performed. However, a fly
with low pteridine levels would hold the increased
levels of pteridines for a longer period of time.
This phenomenon may explain the increased dros op terin
accumulation in the eyes of ras2 flies which had the
lowest levels of drosopterins of any mutant examined
(Brown 1973).
Numerous suggestions have been made with respect
31
to the possible mechanisms of interaction between the
ommochrome and pteridine biosynthetic pathways
Classman (1956) demonstrated that in conversion of
N-formylkynurenine to kynurenine, a formate molecule
is produced for each molecule of kynurenine synthesized.
Lehninger (1970) suggested that this formate is bound
to t e t r ahy drofolate to produce N ^ - f ormyltet rahydro-
folate, which is an important cofactor in the synthesis
°1 purines. Purines produced from the formate molecule
can then be used in pteridine biosynthesis (Albert
1957) by contributing to the pool of one-carbon units
used for purine production as suggested by Letter e t
al_. (19 73). According to this mechanism, feeding
tryptophan or N-formylkynurenine to flies would be
expected to increase levels of all pteridines examined.
The expected results were obtained with tryptophan.
However, treatment with N-f ormylkynurenine did not
give the expected results . The fact that N-formyl
kynurenine did not dissolve in water as did the other
compounds employed may indicate that larvae which were
fed N-formylkynurenine may not have consumed or
assimilated sufficient amounts to elicit a response.
Ghosh and Forrest (1967) assumed that kynurenine-
3-hydroxylase requires a reduced unconjugated pteridine
32
as a cofactor. This assumption was based on the fact
that an enzyme with similar hydroxylation properties,
phenylalanine hydroxylase, requires a tetrahydro-
pteridine cofactor (Kaufman 1959 ; 196 7b). Schwink
(19 70) observed an increase in drosop terin production
in maroon-like and rosy mutants of D. melanogas ter
which had been implanted with phenylalanine crystals
as larvae. These results may be explained by the
following observations. In the conversion of phenyl
alanine to tyrosine, tetrahydrobiopterin is oxidized
to dihydrobiopterin (Kaufman 1959). The dihydro
compound can be converted, non-enzyiatically, to
sepiap terin, biopterin or xanthopterin (Fukishima
19 70 ; Rembold 19 70) . Resulting biopterin may then be
used for drosopterin synthesis. A similar phenomenon
may be involved in the conversion of kynurenine to
3-hydroxykynurenine which, as pointed out, may require
the same co factor as the phenylalanine conversion.
Therefore, an increase in conversion of kynurenine to
3-hydroxykynurenine in an organism could result in
increases in sepiapterin, biopterin, xanthopterin and
drosopterin. Results obtained from the kynurenine
treatment did coincide with the results expected from
the mechanism proposed. The response obtained was an
33
increase in dros opterins, sepiapterin, HB and pre
sumably xanthopterin. Since isoxanthopterin and
xanthopterin were measured together it was not
possible to determine xvhich of these compounds in
creased. The same results were expected for phenyl
alanine fed flies, since it was assumed both con
version of phenylalanine to tyrosine and kynurenine
to 3-hydroxykynurenine require the same cofactor.
However, feeding phenylalanine did not elicite the
expected response in flies. This lack of effect may
have been due to low quantities achieved through
feeding as compared to the levels reached by Schxvink's
(1970) implantations. Another explanation as to
the results of kynurenine and phenylalanine treat
ments is possible. Altered pteridine accumulation
observed by Schwink (1970) and reported here may
have been caused by the products of the reactions,
3-hydroxykynurenine and tyrosine, rather than the
reactions themselves. This explanation may seem even
more feasible since 3-hydroxykynurenine enhanced
pteridine accumulation. However, tyrosine did not
change pteridine accumulation. It seems likely that
an alternate mechanism is involved for 3-hydroxy-
kynurenine. This substance is converted to
34
xanthommatin by the action of phenoxazinone syn
thetase (Phillips and Forrest 1970). Xanthommatin is
gradually reduced to dihydroxanthommatin. It is
dihydroxanthommatin which is associated with the
pigment granules in the eyes (Phillips e_t_ eKL. 19 73) .
Therefore, by feeding large amounts of 3-hydroxy -
kynurenine to IK melaaogaster an increased production
of xanthommatin would result. However, the conver
sion of xanthommatin to its final deposition form
may require a cofactor such as tetrahydrobioptarin.
If this is the case, flies fed 3-hydroxykynurenine
would be expected to show increases in drosopterins,
sepiap terin, xanthopterin and HB as did the kynurenine
fed flies. Results reported here do show increases
in the above p teridines for flies fed 3-hydroxy
kynurenine . Figure 6 shows the points of inter
action between the ommochrome and pteridine pathways
according to the mechanisms proposed.
No explanation can be offered for the large and
consistent decreases in pteridines seen in the testes
of ma-1 mutants.
Since treatments did not have an observable
effect on riboflavin accumulation in the Malpighian
tubes, it may be that the interaction between
Fi gu re 6 . Points of interaction between the ommochrome and pteridine metabolic
pathways according to the mechanisms proposed.
Tryptophan
N-formylkynurenine
-» Formate
Kynurenine
3-hy droxykynurenine
Xanthommatin
— Tetrahydrobiopterin
Dihy d r ob i op te r in
— Te t r ahy d r ob iop te rin
~"*Dihydr ob iopterinDihydroxanthommat in
Purines
7 s 8-Dihydroneop terin
L-Threo-Dihydroneopterin
L-Threo-Dihydrobiopterin
Sepiapterin---->Xanthop terin
Dihydrobiopterin
Biopterin
D r osopterin
37
riboflavin and pteridines is a physical one. Since
riboflavin and pteridines have similar structures,
a fly which is able to accumulate large amounts of
riboflavin may also be able to accumulate and hold
large quantities of pteridines. However, more than
just physical similarities between pteridines and
riboflavin are indicated. For instance, the fact
that riboflavin is used as a cofactor in the pro
duction of isoxanthopterin (Chovnick and Sang 1968)
indicates further relationships between the two
compounds.
It is apparent that some mutants, cl and p n ,
showed less variability in pteridine accumulation
than did other strains examined. That is, the
pteridine levels in c1 and pn were not altered by
any of the treatments. This suggests a new problem.
Are certain strains more "stable" than others? Strains
of mutant D_. melanogas te r have been maintained in
laboratory stocks for years. Obviously a high degree
of inbreeding has resulted. It is possible that some
strains may be able to tolerate a higher degree of
iiiDj_eeding than others. These highly isogenic strains
would then be extremely impliable in experiments such
as these. Another factor which could add to
38
homogeneous pteridine accumulation would be the time
of pigment accumulation in certain strains. It may
be that some mutants accumulate pteridine inter
mediates at times much earlier than wild-type.
Therefore, by the time of assay the levels of
pteridines may have reached a plateau and no alter
ation in levels could be perceived. In addition, the
molecular basis of the mutation may add to the inal-
terable quality of the strain. Althou gh the spe
action s of 'the cl an d pn mut at ions are not known
is pos sible that cer tain pec uliari t;ies o f these
mutati ons may 1e ad to their s tab ili ty . For ins t
a fly with the mutât ion for white eyes (w) does
produc e pteridi ne in termediates at the larval an
pupal s tages. Howe ver, due to the inability of ’
flies to form pi gmen t granul es in the eyes, p ter
are lost comple tely from the organi sm a few days
after eclosion (Zieg 1er 1961 ) . The ref ore , w fli<
would be highly inal terable as far as pteridine
accumu lation is cone erned. The pn and cl mutant:
may be similar to w s trains in that the capacity
vari an ce in pte ridin e accumu lat ion may not be pn
due to the part i cula r point of acti on of the mut
From the results reported here it may be
39
concluded that increases in ommochrome precursor
levels alter pteridine accumulation in jD. melano-
gas ter. The results obtained support the mechanisms
proposed for the interaction. . However, these proposed
mechanisms do not preclude other possible mechariisms
of interaction
V. SUMMARY
Adult wild-type and eye-color mutants (cl, ma-1,
mah , ras^ , 11 , v , cn, st, car, se, cm and pn) of
I), melanogas ter were separately fed ommochrome
precursors, phenylalanine and tyrosine. Chromato
graphic and fluorometrie methods were used to
determine the amounts of certain pteridine inter
mediates in eyes and testes of treated as compared
to control flies. Riboflavin content was examined
in Malpighian tubes.
The eyes of ma-1 , 1t and ^ females showed
significant increases in pteridine accumulations when
fed ommochrome precursors. Certain characteristics
of these three mutant types were realized from the
results of Brown (1973). (1) Adults showed decreased
levels of ommochromes and dr os op t e rins in eyes.
(2) Levels of pteridine intermediates and ommochrome
precursors were reduced in pupae and this reduction
continued through the adult stage. These charac
teristics were considered requisite for flies to
elicite a perceivable response to treatment.
The results reported here give evidence that the
interaction between ommochrome and pteridine pathways
takes place before and during final pigment
41
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