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

L I T E R A T U R E C I T E DAlbert, A., 1957 The transformation of purines into

pteridines. Biochem. J. 6 5 : 124-127 .

Baglioni, C., 1959 Genetic control of tryptophan per­oxidase-oxidase in Pros ophila melanogas ter.Nature 184: 1084-1085.

Baillie, D. L. and A. Chovnick, 1971 Studies of the genetic control of tryptophan pyrrolase in Pros ophila melanogas ter. Mol. Gen. Genet. 112(4) 341-353.

Beadle, G. W., 1937a The inheritance of the color of Malpighian tubes in Drosophila melanogas ter.Am. Naturalist 71: 277-279.

Beadle, G. W., 1937b Development of eye colors inDrosophila: Fat bodies and Malpighian tubes inrelation to diffusable substances. Genetics 2 2 : 587-611.

Beadle, G. W. and B. Ephrussi, 1935 Differenciat ion de la coleur de l'oeil cinnab ar ch e z la Dros ophilae (Pros ophila melanogaster). Compt. Rend. Acad. Sci. 201: 620-622.

Bodine, J. H. and L. R. Fitzgerald, 1948 Changes in riboflavin during embryonic development as a function of the embryo. Physiol. Zool. 21 : 93-100.

Brehme, K. S., 1941 The effect of adult body colormutation upon larvae of Drosophila melanogas ter. Proc. Nat. acad. sci. 2 7 : 254-261.

Brown, R., 1973 Interaction between pteridine and ommochrome biosynthesis in Drosophila melano­gas ter . M. S. Thesis, Creighton University.

Butenandt, A. and G. Neubert, 1958 Über Ommochrom. XVII. Zur Konstitution der Ommine, I. Ann.618: 167-172.

43

Butenandt, A., W. Weidel and H. Schlossberg, 19493-0xykynurenin als cn+ -Gen-abhangiges Glied im intermediären Tryptophanstoffwechsel. Z. Natur­forsch. 4j>_: 242-244.

Cassady, W. K. and R. P. Wagner, 1971 Separation of mitochondrial membranes of Neurospora crass a .I. Localization of L-Kynurenine-3-Hydroxylase.J. Cell Biol. 4_9 (2): 536-541.

Chauhan, N. S. and F. W. Robertson, 1966 Quantitative inheritance of red eye pigment in Drosophila melanogas ter. Genet. Res. J5: 143-164.

Chovnick, A. and J. H. Sang, 1968 The effects of nutritional deficiencies on the maroon-like maternal effect in Drosophila. Genet. Res. 11: 51-61.

Collins, J. G., E. J. Duke and E. Classman, 1970Nutritional control of xanthine dehydrogenase.I. The effect in adult Drosophila melanogas ter of feeding a high protein diet to larvae.Biochim. Biophys . Acta. 20 8 : 294-303.

Danneel, R., 1941 Die Ausfärbung überlebender v- andcn- Drosophila-Augen mit Produkten des Tryptophan­stoffwechsels. Biol. Zentralbl. 6_1_: 388-398.

Danneel, R. and B. Zimmermann, 1954 Uber des Vorkommen und Schicheal des Kynurenine bei verschiedenen Drosophil arassen. Z. Naturforsch 9J>_: 788-792.

Demerec, M., 1965 Biology o f Drosophila, Hafner Publishing Company, New York.

Ephrussi, B. and J. L. Herold, 1944 Studies of eye pigments of Pros ophila. I. Methods of ex­traction and quantitative estimation of the pigment components. Genetics 2_9 : 148-175 .

Fukushima, T., 1970 Biosynthesis of pteridines in the tadpole of the bullfrog Ran a catesbeiana. Arch. Biochem. Biophys. 139(2): 361-369.

Ghosh, D . and H. S. Forrest, 1967 Enzymatic studiesof the hydroxylation of kynurenine in Drosophila melanogas ter. Genetics 55 : 42 3-4 31.

Classman, E ., 1956 Kynurenine formydase in mutants ofDrosophila. Genetics 4 L: 55 6-5 73 .

Classman, E ., 1965 Genetic regulation of xanthinedehydrogenase in Drosophila melanogas ter. -Fed. Proc. 2j4: 1243-1251 .

Green, M. M . , 1949 A study of tryptophan in eye color mutants of Drosophila melanogas ter. Genetics 34 : 564-572.

Green, M. M ., 1952 Mutant isoalleles at the vermilionlocus in Drosophila melanogas ter. Proc. Natl. Acad. Sci. U. S. 38.: 300-305.

Guroff, G . and C. A. Strenkoski, 1966 Biosynthesis of pteridines and of phenylalanine hydroxylase co­factor in cell-free extracts of Pseudomonas species (ATCC 11299a). J . Biol. Chem. 241: 2220-2227.

Hadorn, E. and H. K . Mitchell, 1951 Properties ofmutants of Drosophila melanogas ter and changes during development as revealed by paper chromato­graphy. Proc. Natl. Acad. Sci. U. S. 3 7 : 650-6 65 .

Hadorn, E. and I. Ziegler, 1958 Untersuchungen zur Ent- wicklung, Geschlechtsspeziftat und phanogeneti- scnen Autonomie der Augen-Pterine verschiedener Genotypen. Z. Vererbungsl. 89_: 221-234.

Hendrichs-Hertel, U. and B . Linzen , 1969 Kynurenine-3- hydroxylase in Calliphora erythrocephala. Z .Vgl. Physiol. 6_4_( 4) : 411-431.

Horikawa, M., L . L. Ling and A. S. Fox, 1967 Effects of substrates on gene-controlled enzyme activ­ities in cultured embryonic cells of Pros ophila. Genetics 5_5_: 569-583.

Kaufman, S ., 1959 Studies on the mechanism of enzy­matic conversion of phenylalanine to tyrosine .J . Biol. Chem. 234: 2677-2682.

45

Kaufman, S., 1967a P teridine cofactors. Annu. Rev.Biochem. 36(1): 171-184.

Kaufman, S., 1967b Metabolism.of the phenylalaninehydroxylation cofactor. J. Biol. Chem. 242(17):3934-3943.

Knox, W. E. and A. H. Hehler, 1950 The conversion of tryptophan to kynurenine in liver. I. The coupled tryptophan peroxidase-oxidase system forming formylkynurenine. J . Biol. Chem. 187: 419-430.

Lehninger, A. L . , 1970 Biochemis try, Worth Publishers, Inc. , New York.

Letter, A. A., G . Zombor and J . F . Henderson, 1973 Tryptophan as a source of one-carbon units for purine* biosynthesis de novo. Can. J. Biochem.51: 486-488.

Linds ley, D . L. and E. H. Grell, 1967 Genet i cVariation o f Pros oph i1a me1ano g as te r , Carnegie Institute of Washington, Publication No. 627.

Mainx, F ., 1938 Analyse der Genwirkung durch Faktoren­kombination. Versuche mit den Augenforbenfaktoren von Drosophila melanogas ter . Z. Vererbungsl. 75 :256-276.

Matsubara, M. , S. Kat oh, M. Akino and S. Kaufman, 1966 Sepiapterin reductase. Biochim. Biophys . Acta. 122: 202-203.

McNutt, W . S . and H. S. Forrest, 1958 The incorporation of the cl^ of adenine into a pteridine derivative by Eremothesium ashbyii. J . Am. Chem. Soc. 80: 951-952.

Nickla, H ., 1972 Interaction between pteridine syn­thesis and riboflavin accumulation in Drosophila me. 1 ano gas ter. Can. J . Genet. Cytol. 14 : 105-111.

Nickla, H ., 1973 Maternal age effect associated with yellow pigment in Malpighian tubes of Pros ophila melan ogas ter. Can. J . Genet. Cytol. 1 5 : 437-442.

46

Phillips, J. P. and H. S. Forres t, 19 70 Terminal syn­thesis of xanthommatin in Pros ophila melano g as te r . II. Enzymatic formation of the phenoxazinone nucleus . Biochem. Genetics 4_: 489-497.

Phillips, J . P ., H. S. Forrest and A. D. Kulkarni,1973 Terminal synthesis of xanthommatin in Dros ophi1 a melanogas ter. III. Mutational pleiotropy and pigment granule association of phenoxazinone synthetase. Genetics 7_3 45-56.

Remb old, H . , 19 70 Catabolism of unconjugatedpteridines . p p . 163-178. In : Chemistry and Biology of Pteridines, Proceedings o f the Fourth International Sympos ium on Pteridines. Edited by K. Iwai, M. Akino, M. Goto and Y . Iwanami. International Academic Printing Co., LTD. Tokyo.

Rizki, T. M. and R. M. Rizki, 1963 An inducible enzyme system in larvae cells of Pros ophila melano- gas ter. J . Cell Biol. 17 : 87-92.

Sang, J . H ., 1956 The quantitative nutritional require­ments of Drosophila melanogaster J . Exp. Biol.33 : 45-72.

Schwink, I., 1970 Phenogenic inhibition and enhance­ment of dr os op terin formation in various mutants of Drosophila melanogas ter. Drosophila Information Service 45 : 92.

Shoup, J . R . , 1966 The development of pigment granules in the eyes of wild-type and mutant Pros ophila me 1anogas t e r. J . Cell Biol. 2 9 : 2 2 3-249 .

Stern, C ., 1941a The growth of testes in Pros ophila.I. The relation between vas deferans and testes within various species. J . Exp tl. Zool. 87 : 113-158.

Stern, C ., 1941b The growth of testes in Pros ophila.II. The nature of interspecific differences.J . Exp 11. Zool. 8_7 : 159-180 .

Taira, T ., 1961 Enzymatic reduction of the yellowpigment of Pros ophila. Nature 189: 2 31-2 32 .

47

Tatum, E. L. and A. J . Eaagen-Smit, 1941 Identification of Drosophila v“*" hormone of bacterial origin. J . Biol. Chem. 140 : 575-580 .

Wright , C. P. and E. W. Hanly, 1966 Pteridines in thefat body of a mutant of Drosophila melanogaster. Science 152(3721): 533-535.

Woolf, C. M. , 1968 Principles of Biometry , D . Van- Nostrand, Princeton, New Jersey .

Yen, T. T. and E. Classman, 1967 Electrophoreticvariants of xanthine dehydrogenase in Drosophila melanogas ter. Biochim. Biophys . Acta. 146 :35-44.

Ziegler, I., 1961 Genetic aspects of ommochrome and pterin pigments. Adv. Genet . 10_: 349-403.