Biosynthesis of Serine in '' Neurospora Crassa

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I i 1 I ·j ) ! California State University, Northridge i ; ; BIOSYNTHESIS OF SERINE IN '' NEUROSPORA CRASSA

A thesis submitted in partial satisfaction of the requirement for the degree of Master of Science in Biology by Fereshteh Khosravi Kline /

May, 1973 .----,·----··----·-·----·------·--·---·--···--·------··-··----··-·--·-----1

I I I I I

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The thesis of Fereshteh Khosravi Kline is approved:

Committee Chairman

California State University, Northridge

May, 1973

ii r--~--- l ACKNOWLEDGE~lliNTS

1 ! I wish to extend my sincere appreciation and grat- I I j itude to Dr. Joyce Maxwell for her continuous encourage- :ment, assistance, and guidance on my behalf. Her dedi- 1 !cation and deep personal involvement in the preparation i of this "thesis made this effort possible. I also wish to extend my deep appreciation to Dr. · Bianchi and Dr. Spotts for their participation as members of my graduate committee. My sincere gratitude goes to Sam Muslin and Elaine Leboff for their technical assistance.

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TABLE of CONTENTS

page LIST o:f FIGURES ••••••••••••••••••••••••••••••••••••• vi

LIST o :f TABLES • • • • • • • • • • .• • • • • • • • • • • • • • • • • • • • • • • . • • • • vii

ABST.RACT • ••• • • • • • • • • • • • .. • • • • • • • • • • • • • • • • • • • • • • • • • • • • viii Chapters I. INTRODUCTION •••••••••••••••••••••• , • , •• 1 II. MATERIALS and METHODS •.•••••••••.•••••• 8 Strains ...... Chemicals . .. , ....•...... •... Maintenance and Growth of Neurospora Cultures ...... Genetic Analysis •••••••••••••••••••••• Extraction of Soluble Enzymes ••••••••• En.z yme As says • • • • • •••••••••••••••••••• )-Phosphoglyceric Acid Dehydro genase and Glyceric Acid Dehy- drogenase ••.••.••••••••••••••••• Phosphoserine Transaminase and Serine Transaminase •••••••••••••

Phosphos~rine Phosphatase •••••••

III. RESULTS • ••••••.•••••••• I • • • • • • • • • • • • • • • 17 Genetic Analysis of The Mutant Ser-6 •• Mapping of Ser-6 •••••••.••••.••• Genetic Instability of Ser-6 Mutant • •.•..•.• , •.•.•.•••••••••• Mapping of Ser-6 on Linkage Group V • ••••••••.•••••.• , ••••••• I L__ ····~··

-iv Studies Concerning the Amino Acid Re- quirement of Ser-6 ••••••••••••••••.••• Determination of Serine Re- quirement •.•.••••••.•••••••••••• Growth of Ser-6 as a Function of Serine Concentration •••••••••••• Growth of Ser-6 as a Function of Time on Vogel's Minimal Medium and Minimal Medium Supplemented With Serine •••••••• The Effect of Inoculum Size on Utilization of Glycine by s er-6 ...... a •••••••••••••• A Comparison of Serine Biosynthetic Enzymes in Ser-6 and in the Wild- type Culture ...... , .. . )-Phosphoglyceric Acid Dehydro genase and Glyceric Acid Dehy- drogenase •.••••••.•.•••.•••••••• Phosphoserine Transaminase and Serine Transaminase ••••••.•.•.•• Phosphoserine Phosphatase •••••••

~. DISCUSSION.. • • . • • • • • • • . • • . • . • • • • • . • • • • • • 48 Genetic Instability of Ser-6 ••••.••••• The Nature of the Serine Requirement • C' 6 1n uer- e 1 1 1 1 1 e e • 1 1 e 1 e e • e 1 1 1 1 • e 1 1 e 1 1 1 1 V. BIBLIOGRAPHY. • • • • • • • • • . • • • • • • . • • • • • • • • • • 59

v ---~---~-----l LIST of' FIGURES . I Figure page I 1. Pathways of serine biosynthesis f'rom i 4 ! products of glycolysis •••••••••.•••••••••••••• ' 2. Pathway of serine biosynthesis f'rom gly-

oxylat e IJ •••••••••••••• e • • • • • • • • • • • • • • • • • • • • • • • 7 J. Calculated map of ser-6, his-1 and me-J on linkage group v ..•...••....•...•...•.•.•••• 22 4. Published map of his-1 and me-3 on linkage group V • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. • • • • • • • • 22

5. Growth of' ~er-6 and wild-type Neurospora as a function of serine concentration ••••••••• 27 6. Time course of' growth of ser-6 on serine •••••• 30 7. Glyceric acid dehydrogenase and phospho glyceric aci_d dehydrogenase activities in the wild-type grown on Vogel's minimal medium...... • ...... • . . . . . • . • . • . . . • • .35 8. Glyceric acid dehydrogenase and phosphogly ceric acid dehydrogenase activities in the wild-type grown on Vogel's medium supplemented with serine.. . • . • • . • • • • • • . • • . • • • . • . • . • • 37 9. Glyceric acid dehydrogenase and phosphoglyceric acid dehydrogenase activities in ser-6 •• 39 10. Serine transaminase and phosphoserine trans aminase activities in the wild-type...... 1+1 11. Serine transaminase and phosphoserine trans aminase activities in ser-6 ••••••••••••••••••• 43 _12. Phosphoserine phosphatase activity in the wild-type...... • . . . . . • • . . . . • . • 45 13. Phosphoserine phosphatase activity in ser-6 ••• 47

vi ------·------·l LIST of TABLES I Table page I 1. Linkage data on random spores isolated from the cross: ser-6 X his--1, me-'3...... 20 2. Linkage data on random spores isolated from the cross: ser-6 X his-1, me-3 •••••••••• 21 3. The effect of individual amino acids on the growth of ser-6 and nutritionally ;o·J

wild-type al-2; cot-1 •.••.•••.•. I • I • • • • • • • • • • • 22

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·Vii ,------· ------~------~---·------l ABSTRACT I l I I BIOSYNTHESIS OF SERINE ! I IN NEUROSPORA CRASSA i by Fereshteh Khosravi Kline Master of Science in Biology May 197.3 Two pathways for serine biosynthesis in Neurospora crassa have been reported by Sojka and Garner. The phos phorylated pathway involves )-phosphoglyceric acid, phos phohydroxypyruvic acid, and phosphoserine as intermedi ates, whereas the non-phosphorylated pathway involves glyceric acid and hydroxypyruvic acid as intermediates (1).' The phosphorylated pathway was suggested to be the major pathway of serine biosynthesis in Neurosnora by these authors. In this investigation a serine-requiring mutant

of Neuros:eor~ crassa was mapped on linkage group V and was I studied in order to determine the nature of its biochem- i ical lesion. Each enzyme in the two postulated pathways was extracted from the mutant strain and compared with the extract of the corresponding enzyme from the nutritionally·. wild-type strain of Neurospora crassa from which the mu tant was originally derived. All enzymes involv:ed in the

two path~ays were comparable in activity in the mutant and in the wild-type, indicating that the serine requirement

v.iii ,------·------·------.--·~--·--·--~---·-----, in the mutant does not arise from the loss or alteration I of any of the enzymes in these two pathways.

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ix r------~------... ···--·---- .. ______.. __ ··-··· -··------··--·-_ ------~------1 I INTRODUCTION . .

!serine Biosynthesis I Synthesis of serine in Neurospora crassa has been studied by Sojka and Garner (1). The postulated pathways ' i are diagranuned in figure 1. The enzymes active in the ,Phosphorylated pathway are phosphoglyceric acid dehydro genase, involved in the conversion of 3-phosphoglyceric acid into 3-phosphohydroxypyruvic acid; phosphoserine : transaminase, active in transamination of 3-hydroxypyruvic :acid into 3-phosphoserine; and finally phosphoserine phos ! phatase which is involved in dephosphorylation of phospho- ! I ; serin6 (figure 1). · The enzymes implicated in the non-phos-• 1 · phorylated pathway are glyceric acid dehydrogenase, in : volved in conversion of glyceric acid to hydroxypyruvic 1 acid; and serine transaminase, active in transamination of ; hydroxypyruvic acid into serine (figure 1). Because of ·the higher activity of the enzymes involved in the phos phorylated pathway compared to the non-phosphorylated

pathway under the conditions investigated by these authors, 1 Sojka and Garner suggested that the phosphorylated path way was the major pathway for serine biosynthesis in ' Neurospora crassa. The same two pathways for serine bio . synthesis were demonstrated originally in animal tissues (2, 3). In this case the relative contribution by each I pathway for the synthesis of serine varies from tissue to

1 2

~~~:::~~~Y T~~l~:~~S ~~·~~~~~~e ~!h~:~~-::~:~~:s-~ j in higher plants (5). Studies using isotopic competition ! I land mutants blocked in serine biosynthesis indicated that i !the phosphorylated pathway is the only major source of i serine in E. coli and s. typhimurium (6). More recent studies confirming the phosphorylated pathway as the only significant source of serine were carried out in B. sub tilis (7), M. lysodeikticus (8), D. desulfuricans (9), and H. influenzae (10). A separate pathway leading to serine from glyoxylate has been suggested in a variety of orgru1isms, The postulated pathway is shown in figure 2. The conversion of glyoxylate to serine has been shown to be the major pathway of serine biosynthesis in some animal and pla..nt tissues (1.1. 12, 13). Early studies supported the view that serine precede glycine in the pathway present in bakerts yeast (14), but later studies using short-term isotopic labeling indicated that the pathway from gly oxylic acid to serine predominates in this organism (15). In Neurosnora, Wright reported that a serine-glycine mu• tant studied by her grew better on glyoxylic acid or gly cine than on serine. She suggested that the biqsynthesis of serine in Neurospora involves the conversion of gly oxylic acid to glycine and subsequent conversion of gly cine to serine (16). The pathway suggested by Sojka and Garner clearly contradicts Wright's postulated pathway as Figure 1. Pathways of serine biosJ~thesis from products

. of glycolysis. The "C 1 .. indicated as a product of serine metabolism refers to the single carbon fragment used in :the synthesis of methionine, thymine, and purines.

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,------··--·-···-~------··--·-··----·------·-·-····--·--·------·-·-··------··------~------, I I GLUCOSE I t J l H0l'OCH yH- COOH HOC _ COOH 2 2 H2~H OH OH 3-PHOSPHOGLYCERIC ACID GLYCERIC ACID NAtijfNADH NA~lf NADH

H 0 POCH C -COOH HOCH _c -COOH 2 2 3 2 II II 0 0 3-PHOSPHOHYDROXYPYRUVIC ACID HYDROXYPYRUVIC ACID

lJ transamlna lion I ransaminalionlf

H 0 POCH H _COOH HOCH CH- COOH 2 3 21 2 I NH NH 2 2 3_ PHOSPHOSERINE SERlNE H '' Ci' + CH ._ C 00 H I 2 NH 2 GLYCINE 5

__ ,__ -:.______.______, ______····-·-- .. -- ·-·· .. -· .. ----.. -·"'"" --·-·-···- ·---.. -·----~------, the major source of serine in Neurospora. To support or 1 1 . . I I refute the pathways suggested by Sojka and Garner, mutants 1 I requiring serine were examined to determine whether any of I i . the enzymes utilized in these pathways were deficient or reduced in activity compared to the wild-type. A prelim- : inary study carried out by Sojka indicated that two of the : I four known serineless mutants studied showed no apparent

1 deficiency for any of the enzymes involved in the serine · biosynthetic pathways postulated by Sojka and Garner (17, 18). The other two mutants both showed alterations in

1 the transaminases, although they map on separate genetic sites. Since the mutants studied were isolated from different wild-types, the difference between the enzyme ac- • tivities in the wild-type and the two mutants might have I i resulted from differences in genetic background. To elim-

I • ! ~nate the problem of background diversity, Maxwell iso- : lated five serineless mutants from a single prototrophic • culture (as described in materials and methods). One of these serine-requiring mutants, ser-6 was investigated extensively as described in this thesis. The work in this thesis sought to answer three basic questions:

1. On what chromosome is the ser-6 locus located? 2. What is the nature of the biochemical lesion in ser-6 that results in its serine require ment? J. Does this lesion indicate a major pathway for serine biosynthesis in Neurospora? j Figure 2. Serine biosynthesis from glyoxylate. Glyoxy • late may arise from intermediates in the tricarboxylic 1 acid (TCA) cycle, via the reaction sequence known as the "glyoxylate shunt". Isocitric acid is shunted from the TCA cycle by isocitrate lyase, which splits isocitric : acid to form succinic acid and glyoxylic acid. Al terna- i tively, glyoxyla te may arise from glycolic acid, which is i ' formed from carbohydrate metabolism.

l i l l .. ~ . ··-" ·--··. -· --~- ----·--~ ------·------·------I CITRIC ACID I I HOCHCOOH I I CHCOOH I CH COOH I 2 ISOCITRIC ACID

HOOC .CH CH COOH 2 2 SUCCINIC ACID

HOCH COOH ___.,. OCH_COOH 2 GLYCOLIC ACID GLYOXYLIC ACID

NH3 transamination

NH CH COOH 2 2 GLYCINE -~~ HOCH CHCOOH . 2 I NH 2

SERINE

L--·-·-··--·-·--' I I MATERIALS AND METHODS I !Strains I I A nutritionally wild type strain of Neuros~ora I;crassa, strain C102-15300-4-2A, al-2; cot-1 provided by I . ' I /Mary B. Mitchell was used during these studies. Hereafter :I 1 )this culture will be referred to simply as wild-type. l ; ·, Ser-6 (JBM 4-13) which is used extensively during these i studies was isolated by Dr. Joyce B. Maxwell and Mr. Paul West. Nutritionally wild type conidia from al-2; cot-1 were irradiated with ultraviolet light to twenty per cent survival (two minutes). Serine requiring mutants, includ

ing ~-6, were isolated after filtration enrichment {19). For genetic studies, the strains used were multiple mutant stock B56; KH2{r); P829; ~' bal; acr-2; ~ (FGSC

#1540), C102(t); 37401; Y30549y; C86; ~· cot-1; inos; Xlo-1r nt (FGSC #333), 33933, 37401, Y152 M105, a, J-y:s-1,

inos, his-6 (FGSC #1536), and K744, 361-4, ~· his-1, me-3 (FGSC #780). The phenotypic characters of these loci are described in the table below: Locus PhenotyPe white pigmented conidia stock grows colonially at temperatures above 300 C. bal restricted growth on agar as a hemispheric colony acr-2 acriflavin resistant

8 9

c,aroteno.i_d.___ deiic"fent-ln mycefia;exc-ep"t at--, ~~ low te.mperature · l lnos inositol requiring stock ylo-1 yellow pigmented conidia

Il I nt requires nicotinamide I lys-1 requires lysine I: his-6 requires histidine me-3 methionine requiring stock

, Chemicals 14 Uniformly labeled C o( -ketoglutarate ( 14.3 me/ mmole) was obtained from Amersham Searle Corporation. To assure stability of the labile keto acid, the sample was taken up into steri;te water and dispensed into aliquots containing enough material to carry out single experi ments. The samples were quick frozen in liquid nitrogen and stored in -26° C freezer. Nonlabeled compounds used in these studies were of reagent grade quality. Dowex SOW-XB, 200-400 mesh, used for hydrolysis of hydroxypyruvic acid phosphate and for isolation of glu tamic acid from the phosphoserine transaminase reaction mixture, was obtained-from J. T. Baker Chemical Company. Before use, the reagent was washed with 2N NaOH, 10 ml/gm resin. Then this resin was rinsed with distilled water the eluant pH was approximately neutral. Next the resin was washed with 4N HCl, 10 ml/gm, followed by a dis- ' tilled water rinse. These resins were used in the H+ form. 1.0

,-----·------~------·--·----·-~--··----·------·~- ·------·------·-----· I_ Hydroxypyruvic acid phosphate dimethyl ketal was !hydrolyzed using the following procedure. Twenty mg of salt was dissolved in 2 ml of distilled water. About 0.4 ml ·of dry Dowex 50 in the hydrogen form was added and swirled for 60 seconds. This sqlution was filtered by suction into a small flask. This flask was closed tightly and left in an oven at 40° C for four days. After four days the solution was neutralized using an appropriate amount of sodium bicarbonate. Biogel P 10 was obtained from Bio Rad Laboratories. To prepare beads for the column, the dry beads were stirred into potassium phosphate buffer (pH 8), using 16 ml of liquid for eac;::h gram of dry gel. The beads were allowed to swell overnight. These beads were used to pack the column. The column was equilibrated with po tassium phosphate buffer at 4° C before it was used. The scintillation fluid used was prepared from Spectrafluor PPO-POPOP concentrated liquid scintillator from Amersham Searle. Prepared as directed by the manu

facturer, the scintillation fluid contained 4 gm PPO and 50 mg POPOP per liter of toluene.

Maintenance and Growth of Neurospora Cultures Vegetative cultures were maintained either on agar slants of Horowitz complete medium (20) or Vogel's minimal N (21) supplemented as required by the particular strain used. Stock cultures were kept in a refrigerator 11

~transf~rr;d ..to f'~esh me-diu~-b~f~·re use. ·------! For most growth experiments, cultures were grovm !without shaking at room temperature in 125 ml Erlenmeyer i !flasks containing 20 ml of Vogel's minimal medium Nand !two per cent (w/v) sucrose. Each flask was inoculated )with three drops of conidial suspension. For determina- 1 !tion of dry weight, the mycelial pads were fished from I ithe medium with a spatula after four days of growth, :blotted dry between sheets of' paper towels, and left overnight at room temperature to dry. The cultures used for the extraction of' the enzymes reportedly involved in the synthesis of serine were grown in Vogel's minimal medium containing two per cent (w/v) sucrose. A final concentra~ion of 0.4 mg/ml serine was added for serine requiring cultures. Fifty ml of' this medium was used to wash most of the conidia from a five day old culture grown on 20 ml solid complete medium in a 125 ml Erlenmeyer flask. The conidial suspension was filtered through glass wool before using it to inoculate 700 ml of Vogel's minimal medium contained in a low form culture flask. These cultures were harvested after forty eight hours growth on a shaker bath at 25° c.

Genetic Analysis All crosses were done on Westergaard-Mitchell me (22) containing two per cent (w/v) sucrose and sup- as required by strains used ln the cross. -·------~----·-----·---·---·--··------·---, Crosses were prepared by coinoculation of agar plates with 1 appropriate conidia.

Extraction of Soluble Enzymes Mycelia were harvested on a Buchner funnel with suction. The pads were blotted dry between sheets of paper towels, These pads were weighed and then ground with washed sea sand in an ice cold mortar and pestle. Glyceric acid dehydrogenase and phosphoglyceric 'acid dehydrogenase were extracted from the broken cells with five volumes of 0,01 M Tris-HCl, pH 7.5. The crude extracts were centrifuged at 12,350 g for 15 minutes. The supernatant then was centrifuged at 100,000 g for one hour in a model-L ultracentrifuge. This supernatant was assayed for dehydrogenase activity. Phosphoserine phosphatase was extracted from ground mycelium with five to ten volumes of 0.01 M Tris-HCl, pH 7.5. The crude extracts were centrifuged at 12,350 g for 15 minutes. The supernatant was precipitated with solid (NH4)2 S04 added to seventy per cent saturation. This solution was left on ice for 30 minutes and then centri fuged at 12,350 g for 15 minutes. The pellet was rinsed with buffer and suspended in 2 ml of 0.01 M Tris-HCl, pH 7.5. Phosphoserine transaminase and serine transaminase were extracted from ground mycelium with five volumes of 0.01 M potassium phosphate buffer, pH 8, The mixture 1}

c-;;~t;.i.fug;d-;;.t -i-2;).56 g f~r i5 ;..i_;:;,;:;;;,-;:-- Tb.-;-.;;;:pe-rna t.m:;;----~

was then chromatographed on a Biogel P 10 column (2.0 em ,~ x 17 em) at 4° C. The protein peak eluted with 0. 01 M I

potassium phosphate bu~~er was used for assay.

Enzyme As says I I l-Phosphoglyceric Acid Dehydrogenase and Glyceric Acid Dehydrogenase

Activity o~ 3-phosphoglyceric acid dehydrogenase

was measured by the disappearance o~ reduced nicotinamide adenine dinucleotide (NADH) according to the method of Umbarger et al. (6). D-Glyceric acid dehydrogenase was ' [ -- : assayed by replacing hydroxypyruvic acid for phosphohy- , ' droxypyruvic in the reaction mixture. The reaction mix

, ture contained 600 ~ moles of Tris-HCl buf~er (pH 8. 7),

• 4 I' moles of' MgC1 , 0.4 ft moles o~ NADH, and 0.6 fo moles i 2 i of substrate in a. volume o~ 3.2 ml. Approximately 1.0 mg

; o~ protein from a crude extract was used in the reaction . mixture. Protein concentration was determined using the i Biuret test (23). Oxidation of NADH was followed spectro ' photometrically in a Beckman DB-G recording spectrophoto- ·

meter at 340 m~ • Results are shown in fl mole . NADH oxi

dized per mg protein per hour in the presence o~ added substrate minus the background activity in the same units.

Phosphoserine Transaminase and Serine Transaminase Phosphoserine transaminase and serine transaminase were assayed using Sojka and Garner's method (1). The !

'-- ... -.. -----·------~,-·' Ir::------·------·------~------1reaction was carried out in the reverse of the serine bio- ' synthetic direction with either L-serine or L-phospho- 14 i serine added as the amino donor, and c ell-ketoglutarate I I as tl'1e amino acceptor. The amount of labeled glutamic I lacid formed in this reaction was used as a measure of I i t . . l ransam~nat~on. The reaction mixture contained 500 I ' 1 moles potassium phosphate buffer, pH 8; 20 p moles L-

.: serine (or 40 11 moles DL-0 phosphoserine); 20 mg pyridoxal ' 14 phosphate; and 0.06 ~moles of C «-ketoglutarate {spe- 7 ;cific activity 3.14 x 10 c.p.m/)(mole). This mixture !was incubated at 26° C for five minutes before the reac-

; 1 tion was begun by adding about 5 mg of protein from a ;crude extract that had been passed through a Biogel P10 i ·column_ Total volume of the reaction mixture was 5 ml. The reaction was stopped by adding a one ml aliquot of the reaction mixture to 0.1 ml of trichloroacetic acid. i Samples were taken at different time intervals. The pro tein precipitated by the trichloroacetic acid was pelleted I by centrifugation. A 0.75 ml sample of the supernatant was applied to a small Dowex 50 W-X 8, 200-400 mesh, col- . + umn ~n H form. This column was then rinsed with 20 ml of distilled water. Amino acid was eluted with 8 ml 6N

NH40H. These collected samples were evaporated to dryness in a vacuum desiccator. The dried sample was dissolved in I ; 0.35 ml of distilled water. A 0.1 ml sample of this so- l lution was applied to a small circle of Whatman #2 filter : paper and evaporated to dryness in front of a heat lamp. 15 r------·-···-·------·----·--· ... -··-·------·------.. ____ .. ______-·- --· ---·---- ... --- -.- ·-·-·-- - 1 The paper was put into a scintillation v-ia.T-;·-io-mi oF·--·----~ scintillation fluid was added, and the sample was counted j

in a liquid scintillation counter, model OJT004A Kobe In- 1 dustrial Corporation. Enzyme activity is expressed as

m~ moles glutamic acid formed per mg protein per hour.

The relationship between c.p.m. and~ moles glutamic acid was estimated by using known amount of the uniformly la-

; beled ~-ketoglutaric acid precursor and determining the c.p.m. obtained under the conditions of this experiment.

Phosphoserine Phosphatase Phosphoserine phosphatase was assayed by determin : ing the amount of inorganic phosphate formed from phos- : phoserine in the presence of enzyme by the method de scribed by Ames (24). The reaction mixture contained 200 ;){ moles of Tris-HCl, pH 7. 5, 50 tt moles Mg Cl2, 20 /<- moles of DL- phosphoserine, and approximately 1. 5 mg of protein. in a final volume of 4 ml. The reaction mixture was incu : bated at 26° C b~fore the reaction was begun. The reac tion was started by addition of protein. This reaction was stopped by adding 0.7 ml of the reaction mixture to 2.3 ml phosphatase mix (the mix contains one part 10% ascorbic acid ru1d six parts 0.42 per cent ammonium mo-

lybdate. 4H20 in 1 N H2S04). This solution was incubated • for 20 minutes at 45° c. The absorbance of the mixture

was read at 800 m~ in a Beckman DB-G spectrophotometer.

Enzyme activity is expressed as~ moles phosphate formed 16 r-·------"·-----···------.. --- ...... -----· .... -·- .... _- ·------.. -· per mg protein per hour. Genetic Analysis of The Mutant Ser-6

Mapping of Ser-6

To determine the location of ser-6, the mutant was

,crossed with two strains; B56; Kh2 (r); P829; ~; bal; . acr-2; we and C102; 37401; Y305J9y; C86; a, cot-1; inos; ylo-1; nt. Random spores from these crosses were spread . on four per cent agar plates. Individual spores were iso ;lated onto small slants containing supplemented medium. • These cultures were heat shocked by placing the tubes in ! a 60° C water-bath for about one hour. Preliminary results from these crosses indicated that ser-6 might be on , linkage group v. To further locate the locus on linkage :group V, ser-6 was crossed with lys-1; inos; his-6. Be- ' 'cause of the peculiar results of this cross which are de : scribed below, another cross was performed between ser-6 • and his-1, me-J.

Genetic Instability of the Ser-6 Mutant

During the analysis of the cross invo.Lv~ng ser-6 i and lys-1, inos, his-6, the frequency of wild-type progeny I ' far exceeded the expected value based on reciprocal progeny. At first, this feature was considered to be the re sult of either contamination of crosses by a wild-type Neurospora or contamination during transfer of conidia to different media due to poor technique. But several related

17 f"observat-i0.i18--ieci_t_o_ ·z.e-..:evaiU:ation-·oi ___th.18 ___1ligii--wTICi:-=t-Y.Pe -·--1 ~~requency as a property o~ mutant ser-6 itsel~. Fresh I !crosses o~ ser-6 and lys-1, inos, his-6 performed with I l ! jcareful attention to prevent contamination still showed l !a high frequency of wild-type. Also other individuals I [independently evaluated this cross and obtained similar ·results. Poor technique in transferring was eliminated :as the responsible factor. A separate cross involving ser-6 and his-1, me-3 again showed a disproportionately ·high frequency of wild-type. An attempt to find a pattern. ! to the results which might explain the cause led to the ; observation that a higher frequency of wild-type progeny was obtained from crosses involving ser-6 when spore isolates were analysed later than five to seven days after . germination. Also in the case of vegetative reisolates

of the original ser-~i mutant reversion to wild-type would occur upor1 standing. If conidia from these wild-type re . vertants were suspended in water and spread on serine- supplemented minimal agar plates and incubated for several days, it was possible to pick up individual serine-requir ing colonies. These observations suggest that ser-6 has a high frequency of spontaneous reversion.

Mapping of ~i on Linkage Group V

Most of the data from the cross involving ser-6 and lys-1, inos, his-6 were collected when the cause of the high number of phenotypically wild-type progeny was unknown. Large numbers of progeny were transferred when the 19

..,------~------·-·-·-··--···---···-···--·-··········-~-···-··--·····-··------, isolates were over seven days old. These data are not 1

Jused for mapping the ser-6 locus, The data from ser-6 and j lhis-1, ~e-3 crosses can be seen in table 1. From these l ' ! i jdata only those results obtained from spores transferred j :between one to five days after germination are used for !mapping ser-6. These data are given in table 2. The cal- ' i culated map is shown on page 22.• ~-·------.. -•·-"~""-•-•~·--·~ --· ••- • •~ ••---••• .-,.---·---·---••w••·~--·-••·-••••- •••••-···~--, ... ,_,.,,~---•• ·-·····-·-···-···--·-···-·····--·-· ...... - .... - ...... _.. ______,

I iTable 1. Linkage data on random spores isolated from the cross: ser-6 X his-1, me-3 I 1 These data were collected from progeny transferred between one to fifteen days after :spore germination. ------1 recombination I I zygote parental single single double total I genotype combination region I region II region I+II %germination! I ------1I his-1 me-3 + 255 10 5 2 549 - I + + ser-6 201 29 45 2 89-95 I ! region I region II ------1

!\) 9

. ·----~-~--·---·------·-- ·········---~--- . '. ----·-·-·------··· ···--.. - ··--·· I I l I .Table 2. Linkage data on random spores isolated from the cross: ser-6 X his-1, me-3. 'These data were collected from progeny transferred between one to five days after germ :ination.

recombination I zygote parental singie single double total . 1 genotype combination region I region II region I+II %germination 1

his-1 me-3 + 108 3 2 0 254 I - + + ser-6 120 13 7 1 89-95 region I region II ! ------~~------:______~::~-- I I I

_____ .J ·-···-----~~---~-----~~-"--··----·------·--·-·-"···-----~-~- ., ______·------.. --~----· ------·· ·- ·-·· -- -~------·-·-··· ------·------I

N !-'--" 22

r------~--··--···--·-··-~ -~----·-·--·-·--·------·- -·---·------~----- ·-·----~---·-·-.. ------·------~ I ! Figure 3. Calculated map based on table 2.

his-1 ser-6 ._____ 6.6

.______.....~ 10.6 ,______

Figure 4. Published map (25).

me-3

~------10.5 ------.J 23

---~------··----·------·----··------·------·------l

Studies Concerning the Amino Acid Requirement of Ser-6 !

Determination of Serine Requirement ! I 1 I The mutant ser-6 was isolated by a technique de- 1 I • d [ !s~gne to select serine auxotrophs. To confirm that jserine was the only amino acid to which ser-6 would re- I l ispond, seventeen amino acids were tested individually. The result shown in table J, indicated that serine is the only single amino acid which permits significant growth.

Growth of Ser-6 as a Function of Serine Concen- tration As shown in table 3, serine at a final concentra tion of 0.1 mg/ml does not allow ser-6 to grow as well as the wild-type. This result.suggests that ser-6 might re quire more than 0.1 mg/ml of serine for optimum growth. The results shown in figure 5t indicates that optimum growth of ser-6 is obtained at a final concentration of 0.5 mg/ml of serine. This amount of serine allows the mutant to grow to the same extent as the wild-type in three days.

Growth of Ser-6 as a Function of Time on Vogel's Minimal Medium and J.Vlin~mal Med~um Supplemented with Serine Dr. Maxwell (18) observed that a serine requiring ; mutant, DW-110, grows with the same rate as wild-type after a four day lag on minimal medium, whereas growth of the wild-type or the mutant in the presence of serine begins ,------·------·------·------·------·------,

~ABLE 3. I The effect of individual amino acids on the growth ! of ser-6 and nutritionally wild-type al-2; cot-1.

' w~ld-type ser-6 1 Added amino acid al-2; cot-1 mg dry wt mg dry wt none 54.6 trace 'DL-alanine 56.8 trace : L-arginine 62.2 trace L-aspartic acid 50.9 trace

1 L-cysteine 45.7 trace ; L-cystine 36.3 trace ; glycine 22.9 trace · L-histidine 42.9 trace L-isoleucine + L-valine 51.3 trace i L-leucine 50.0 trace ; : L-lysine 49.6 trace ; L-rnethionine 44.5 trace : L-phenylalanine 47.9 trace ! L-proline 55.3 trace I ! L-serine 18.5 i 43.2 l DL-threonine 41.6 trace i L-tryptophan 37.4 trace L-tyrosine 53.0 trace ! • • : L-val~ne 33.2 trace i casamino acids 61.9 trace 25

r·-~.. ··---.-- .. ·------~·------~-·-·---··~~. -· ~ --·-·---~-~- ·-·----~ .. ·-- -~.------· --~------~---- -l 3 ~~TABLE ~u~:::::n:::: grown stationary ~or three days in 20 ml Vogel's minimal medium in 125 ml Erlenmeyer flasks at I ! 25° c. Amino acids were added to give a final concentra- \ ) !tion of 0.1 mg/ml for L-amino acids, and 0.2 mg/ml for l DL-amino acids. All dry weights are averages of tripli- i : cate pads. 26

:Figure 5. Growth of ser-6 and wild-type al-2; cot-1 as ·a function of L-serine concentration. Cultures were grown :in 125 ml Erlenmeyer flasks containing 20 ml Vogel's med ; ium and two per cent (w/v) sucrose. L-serine was added to a final concentration which ranged from 0.1 mg/ml to 1e0 • mg/ml. Flasks were left stationary at room temperature i . for three days. All dry weights are averages of duplicate ·

i pads. I I

i l ! ! 2{.

~------·------·--·-·-·

I I

- 70 - .... 0

• •WILD TYPE

oo--0 SER.6

0.2 0.4 o.6 0.8 1.0

L.SER (m9/ml) 28

at two --d:;;.y;:--·Totest "W"h-~th:;r-·s;r.·:K'"l)-eilaves--similar ly ,-- growth of the mutant as a function of time was studied. The results indicated that ser-6 does not grow on Vogel's minimal medium even up to six days after inoculation. The results of growth on Vogel's minimal medium supplemented with serine are shown in figure 6. These results indicate that the growth of ser-6 on minimal medium supplemented with serine is negligible up to twenty four hours. By three days this growth reaches half of the maximum level which is attained at four days.

The Effect of Inoculum Size on Utilization of Gly cine by Ser-b

observed that in several serine requiring mutants, growth on 0.5 ~ mole of glycine at five days shows a linear re lationship with inoculum size. To test whether the same phenomenon would be observed with ser-6, this mutant was grown on 0. 5 )'f. mole of glycine with variable inoculum size•. The result of this experiment indicates that ser-6 cannot utilize glycine for growth regardless of the inoculum size. 29

r------·----·-·- ·-···~-·---·--·---·----··

·Figure 6. Time course of growth of ser-6 on L-serine. I i Cultures were grown on 20 ml Vogel's medium and two per

1 cent (w/v) sucrose in 125 ml Erlenmeyer flasks. Serine was added to give a final concentration of 0.4 mg/ml. Triplicate cultures were harvested and their dry weight 1 determined at one. to six days after inoculation. 50 -

0 0 40 -

-.,tit E -Q 30 - c( D. u. 0 ...::z:: 20 - ~ loll ~ > ~ 10 - Q

5 6

TIME AFTER INOCULATION (days)

l . -·--·~----~-'-"' Jl

------··------·--·----·--- --··----·---·----~------, A Comparison of Serine Biosynthetic Enzymes in Ser-6 and I Wlld-tlEe Culture ' I~ An attempt was made to identify the location of the ' !altered enzyme in ser-6 which would account for its serine: !requirement. All enzymes in the two pathways described by l !Sojka and Garner (1) were extracted from ser-6 and the ! lwild-type and their activities were compared. The ex- t !traction and assay of each enzyme was carried out as de .i ! scribed by Sojka and Garner.

, 3-Phosphoglyceric Acid Dehydrogenase and Glyceric jAcid Dehydrogenase The first enzymatic step in the two parallel path ways involves phosphoglyceric acid dehydrogenase or gly ceric acid dehydrogenase. The activity of these enzyrrtes were compared in ser-6 and the wild-type. The results ob tained can be seen in figure 7, figure 8, and figure 9. These results indicate that neither wild-type nor ser-6 when grown on Vogel's minimal medium containing sucrose and supplemented with serine in the case of ser-6 shows any demonstrable activity for glyceric acid dehydrogenase. The phosphoglyceric acid dehydrogenase activity of the two cultures grown in Vogel's medium containing sucrose and supplemented with 0,4 mg/ml serine do not differ signifi cantly from one another. The specific activity of phosphoglyceric acid dehydrogenase in ser-6 corrected for the background activity as measured by a control containing no

hydroxypyruvic acid or phosphohydroxypruvic acid is 2.0 ~ .3-2

moles-of"·-·N:Anli oxid.iz ed.---p-er -·-mg_o_t_p_ro:Eein :Perli~u-rcomp-a:r-ed.-J to 1, 70 I" moles of NADH oxidized per mg of protein per houri in the wild-type. The speci~ic activity o~ the wild-type 1 ' ! grown on Vogel's medium containing sucrose but no serine is 1.10 ~ moles o~ NADH oxidized per mg o~ protein per hour. This result indicates that the presence o~ serine does not inhibit the activity o~ phosphoglyceric acid de hydrogenase.

Phosphoserine Transaminase and Serine Transaminase

As shown in ~igure 10, and ~igure 11, very little phosphoserine transaminase activity is demonstrable in the wild-type or ser-6 within 25 minutes. However; the phos phoserine transaminase activity is equal to serine tran saminase activity by 60 minutes in both the wild-type and ser-6. For the purpose o~ comparing the two cultures, speci~ic activity with each substrate has been estimated

~rom the readings at ten minutes. The speci~ic activity -3 o~ serine transaminase in ser-6 is 3.48 X 10 fi moles/mg/ hour as compared to 2. 57 X 10-3 J4- moles/mg/hour in the wild-; i type. From the summarized results in ~igure 10 and ~igure I i 11 and ~rom a comparison o~ the speci~ic activities, it 1 I may be concluded that ser-6 and the wild-type do not di~fer' ! significantly in the enzyme activities measured under the conditions of this experiment.

Phosphoserine Phosphatase The last enzyme investigated was phosphoserine 33

·--·---~-~------~---·------·----

phosphatase. The summarized results o~ the assay are pr~- II

sented in figure 12 and ~igure 13. The activity o~ the I enzyme is 5.54.11- moles o~ phosphate/mg/ho~r ~or ~r-.2_ com- I i I 1 1 pared to 2.40~ moles o~ phosphate/mg/hour ~or the wild- I 1 type. As shown in ~igure 12, ~igure 13, and also by a I !comparison o£ the respective activities, ser-6 does not ! iSeem to be de~icient ~or phosphatase activity under these I jexperimental conditions. i i 1 In summary, no major di~~erences were demonstrable :between comparable serine biosynthetic enzymes activities in ser-6 and the wild-type.

I I L...... _-·- ...... ··- -·-· --- Figure 7. Glyceric acid dehydrogenase (G.A.D.) and_phos ; phoglyceric acid dehydrogenase (P.G.A.D.) activities in : the wild-type grown on Vogel's minimal medium. Enzyme activity is measured by disappearance of reduced nicotina mide adenine dinucleotide (NADH). Oxidation of NADH was followed spectrophotometrically at 340 m I" • The reaction ; mixture contained approximately 1.0 mg of protein from a i crude mycelial extract. ! I

0.6 -

i... e 0.7 - c ~ M

Q . d

o--o P.G.A.D. 0.8 • • G.A.D. o---o CONTROL

2 4 6 8 10

Tl ME (minutes) Figure 8. Glyceric acid dehydrogenase (G.A.D.) and phos i phoglyceric acid dehydrogenase (P.G.A.D.) activities in the wild-type grown on Vogel's minimal medium supplemented

; with serine to a final concentration of 0.4 mg/ml. Enzyme 'activity is measured by disappearance of reduced nicotin- , amide adenine dinucleotide (NADH). Oxidation of NADH was

followed spectrophotometrically at )40 m fi . The reaction ; 1 I mixture contained· approximately 1.0 mg of protein from a , crude mycelial extract. ,------~------

1 - I

( • e Q 0.7 - =~ M Q• • 0

0 0 P. G.A.D. o.s - • • G.A.D. 0 0 CONTROL -I 2 4 6 8 10

TIME ( minutes ) J8

r-·----·---~---··--~------·---· .. ··-.. ____ .. _ ·-··--········------~--

1 !

I I I i'

Figure 9. Glyceric acid dehydrogenase (G.A.D.) and phos phoglyceric acid dehydrogenase (P.G.A.D.) activities in ser-6 grown on Vogel's minimal medium supplemented with :serine to a final concentration of 0.4 mg/ml. Enzyme • activity is measured by disappearance of reduced nicotinamide adenine dinucleotide (NADH). Oxidation of NADH was , i . followed spectrophotometrically at 340 m /f. • The reaction !

'mixture contained approximately 1.0 mg of protein from a · crude mycelial extract. 39

--_____.,.... h~-- ~- -· ~-·----~·---·---- ·-----~- ·------:

0.6 -

~e 0-7 =~ M c• 0.

8 & P.G.A.D. ••--.,.• G.A.D. 0.8 - oo---oo CONTROL

2 4 6 8 10

TIME (minutes) ------.------·-·-·--l

Figure 10. Serine transaminase and phosphoserine transam

inase activities in the wild-type. Enzyme activity is ·.j measured by the formation of c14-glutamic acid by tran samination of c14 ~-ketoglutaric acid in the presence of enzyme and serine or phosphoserine. The radioactive glu tamic acid product was isolated on a small Dowex column and counted. Each point on the graph represents the a mount of glutamic acid produced in the reaction mixture containing approximately 5 mg protein. Results from two separate experiments have been combined to construct the curves. Points obtained from one experiment are indicated by ftoo-~o , e---.t> , and ·~---1'1:~. Points obtained from a second experiment are indicated by •'----•••------~· and .,., ..

. I ·I

G) -..as w 0 z -.Q :::J a:- 0 =c.r:> •• w 0 en :::J 0 .: wcn&:~ zoa:0 ~:x:,_ wa..z =U") en..... 'o .. ..JCO ~ G) l ] l ~ ~ = c:::J ·-E ~ w :i: -J- z 0 -tc co ::;) 0z

-I

-I M - I Figure 11. Serine transaminase and phosphoserine transam inase in ser-6. Enzyme activity is measured by the forma tion of c14-glutamic acid by transamination of c14 ~-ketoglutaric acid. The radioactive glutamic acid product was isolated on a small Dowex column and counted. Each point on the graph represents the amount of glutamic acid produced in the reaction mixture containing approximately I I 5 mg of protein. Results from two separate experiments I have been combined to construct the curves. Points ob- I tained from one experiment are indicated by o----o•~--~·' and*~---4~· Points obtained from a second experiment are indicated by •'--••, ____., and "ifl"•--..•· ,... -..a i Ul ..Q-"' z :1 ! De "' -~= I Ul :1 l V) c"'

I 0 ·- I :c-5. i .... a.. ... : ' z"'O-0~ I Gii::CI- wa.z -fg "l.Jo ...10U • ~ • ---"' ] ] ":1 -II: l - c -=t ._,·e Ul ~ ..... z -M= 0 .....-

N -

; -·--··· ·~---···-·~-~----·---·~------~---"·"--.,--. _,__..., I I

. Figure 1). Phosphoserine phosphatase activity in ser-6.

The reaction mixture contained 2001' moles of Tris-HCl

(pH 7.5), 50}'(. moles of IVIgC1 2, 10/" moles of phosphoserine, and approximately 1.5 mg of protein, in a final volume of • 4 ml. Phosphoserine was omitted from one control and pro- •

tein from another. The reactio~ was started by the addi tion of the protein. At 5, 10 and 20 minutes. 0.7 ml aliquots were removed and assayed for inorganic phosphate. The non-phosphoserine containing control gave a reading i below zero • 45

COMPLETE MINUS PROTEIN

.08 - -... tn Cl) 0 -06 - E ...... _, 1.&1' ~ :X: .04 - A. V2 0 :X: A. .02

5 10 15 20

TIME (minutes) 46

I !

I I ! Figure 12. Phosphoserine phosphatase activity in the wild- type. The reaction mixture contained 200je moles of Tris- , HCl (pH 7. 5), 50 I' moles of 1VIgCl2, 10 )" moles of phospho-

j serine, and approximately 1.5 mg of protein, in a final !volume of 4 ml. Phosphoserine was omitted from one con- . trol and protein from another. The reaction was started ·by the addition of the protein. At 5, 10 and 20 minutes, ; 0.7 ml aliquots were removed and assayed for inorganic , phosphate. The non-phosphoserine containing control gave . a reading below zero.

j ~ • _, --·.-•·~ • ·-•·• ·•- , ... , •• •• ~-·---···-···-L) MINUS PROTEIN

.12

.to -

0 -CD 0 E .08 ___,~ ..... ~ :t: .06 A. Vj i 0:z:: D. .04

-02

5 10 15 20

TIME ( {'ninufes) ,r·--·--·----·--·-·-···-----·-·-----·------·--·-·------··-··-·--··------·----·--- I I DISCUSSION Ii Gene +"... 1.c Instability of' Ser-6

I A high degree of' spontaneous reversion in vegeta- ! · tive cultures of' ser-6 was observed during this study. Similar phenomena have been observed by other investi gators working with Neurospora. Giles in a study con- :cerning the rate of' spontaneous reversion in inositolless mutants of' Neurospora crassa observed that one inositol allele called JH5202 reverted about a thousand times more : frequently than most of' the other alleles which showed a f !uniformly low (0.02-0.01 per million) degree of' revert- ! ibility (26). A high degree of' revertibility was also .observed by Barnett and De Serres (27) in a study involving a Neurospora mutant, number 137 of' the adenine re-

~quiring mutant, ad-3B. The ad-3B (number 137) allele re verted to a phenotypically wild-type allele with a fre 2 quency of three to f'our per 106 conidia, which is 10 to 104 times more frequent than the reversion at other known ad-3 mutant sites. The apparent wild-type obtained by reversion of' ad-3B (number 137) also showed a high degree of' instability signified by mutation back to ad-2B (number : 137) f'rom which they originally arose. Based on their observations, Barnett and De Serres concluded that the ad-JB (number 137) site had become very susceptible to mechanisms of' spontaneous mutation that cause this site

48 rt~-~s~i t~h·-·b~ck-~d forth bet~-;-~;--two -~nstabl~--;.ii~li~--l forms. Unfortunately, very little has been reported about

mech~~isms that could account for this kind of spontaneous

instability. The suggestion made by Barnett and De Serres~ to explain the instability in ad-3B (number 137) is that the original mutation that gave rise to ad-3B (number 137)

from wild-type was a transversion (AT~,--~~G) and the in- . stability observed is restricted to complementary tran ' si tional changes (TM------.CG). If this b.e the case, the two ; unstable alleles would rarely mutate back to stable state, because it would require a transversion. But each unstable form could mutate spontaneously by transitional change to the other at a high frequency. Their model re quires that one of the two unstable alleles permits the i i I synthesis of a fu~ctional gene product. This model could

1 be tested using mutagens that enhance transversion or I 1 transition. Bausum in a recent study observed a high prote- I trophic frequency caused by meiotic reversion in crosses I involving certain alleles of an isoleucine-valine (iv-1) mutant (28). Crosses involving ser-6 also show a higher prototrophic frequency than expected, but whether this high frequency is explained solely by vegetative reversion or whether meiotic reversion may be involved as well is still unknown. 50

1-The--Na::~:::e~:::: :::::::::::;::s::::~:::-:l I the nature of the biochemical lesion in ser-6 which re lsulted in its serine requirement. I I Sojka and Garner had published evidence for two j I !pathways of serine biosynthesis in Neurospora (1). If ! ! these pathways were the major pathways of serine biosyn-

J thesis in Neurospora, it seemed reasonable that one of the i • enzymes involved in one or both of these pathways would :be defective in ser-6. Each of the enzymes involved in ! the published pathways was prepared from ser-6 and its ac-: tivity compared to that of the comparable enzyme from the

wild-type. No majo~ differences were observed between the activities of these enzymes in ser-6 and the wild-type that could account for the serine requirement of the ser-6 mutant. Sojka investigated the serine biosynthetic enzymes

in the three serine-requiring mutants which were available I at the time he carried out his study. Both ser-2 and ser-J showed ten per cent of the wild-type activity of

phosphoserine transaminase. Mutant ser-2 showed three ) I times the wild-type activity for serine transaminase, while; ! ger-1 showed a reduction in serine transaminase activity (17). Under the reaction conditions used in this study

and Sojka's study, the transaminase activity is rather low 1 {in this paper negligible for phosphoserine transaminase, 3 serine transaminase activity was 2.57 X 10 ~ moles -·--~~---- ·-- ,.-~-3 51

--.. ·------·-·-· ----·····-··-·-·-- -~--· -·---·-----·-······ ···------···· .... ·--·~------·------, glutamic acid formed/mg protein/hour in al-2; cot-1 wild- j

type). It should be noted that the three wild-type strains~

investigated by Sojka, Maxwell and this investigator show. 1 significant differences in transaminase activities. Li-1 \ -2 ! A has phosphoserine transaminase activity of ).04 X 10 fi -2 moles/mg/h. and serine transaminase activity of 2.75 X 10 I _x moles/mg/h .. (17).. Strain ST74 A investigated by Maxwell' shows phosphoserine transaminase activity of 7,.91 X 103 ~ moles/mg/h. and no significant serine transaminase activ ity (18). The culture al-2r cot-1 studied in this thesis shows serine transaminase activity of 2.57 X 103 ~ moles/ mg/h. and no significant phosphoserine transaminase activ ity. The mutants investigated by Sojka came from diver gent backgrounds. Sojka tried to reduce the effect of heterogeneity in the genetic background of the mutants by crosses made to wild-type Li-1 A (17), but it is possible that this effort was not completely successful. It is also unknown whether phosphoserine transaminase or serine transaminase activity is due to a specific transaminase or to a general transaminase (1). Sojka found no differ ences in the specific activity of any of the serine bio synthetic pathway enzymes in either H605 ser-1 or 0127 ser-1 compared to wild-type (17). Another serineless mu tant studied by Maxwell, ser-4 (called P110 in her thesis and later changed to DW-110 by the original isolator) also i !did not show any alteration of any of the enxymes involved I lin the published serine biosynthetic pathways. Thus ser-1 52

r=------.. -·------·· ····---·--·"'"'"'-•>-• - ···--·- ...... _ _. .. ---'-· .... - ...... ______. ___ ...... --.. ---·-·---·-·-~------, and ser-4 are similar to ser-6 with respect to the wild- I 1 1 type activities observed for each of the enzymes indicated j lin serine biosynthesis in the pathways described by Sojka I I' and Garner. Each of these mutants maps on a different ' /linkage group indicating that the three mutants involve i separate genes. ' Wagner et al. observed a group of Neurospora iso- ; leucine-valine requiring mutants which did not show any

:apparent de~iciency in the enzymes involved in isoleucine ,valine biosynthesis (29, 30). In their study enzyme ex-

j tracted from the mutant mycelium showed a high activity compared to the wild-type, despite the evidence from ac

cumulation of inter~ediates and feeding experiments that , the enzymes were inactive in vivo. They explained this :phenomenon by suggesting that the enzyme activity within the cell requires a particular arrangement of the isoleucine-valine biosynthetic enzymes within an aggregate. The mutants are postulated to be organizational mutants in the sense that they are able to produce the necessary complement of enzymes, but are unable to organize them in vivo for an effective synthesis of the amino acids. The importance of organization of enzymes has been emphasized as well by the work of Lynen. This investi gator studied the mechanism of fatty acid SJmthesis from

i malonyl CoA using enzyme extracts from yeast (31). He i suggested that the sequence of the reaction was accom plished by a multienzyme complex. The functional unit of 53

_,_. ~·~~----·------_...... -...... ---~-~·~----~------, this complex was proposed to consist of sev~n -d~i-f-f-er-~~t l enzymes arranged around a central sulfhydryl group. Re peated attempts to split the multienzyme complex of yeast into its subunits with retention of the individual enzyme 1 I I !activities were without ·success (31). I, I I Whether the explanation for the serine requirement I i lor ser-6 mutant involves organization of serine biosyn- 1 - i )thetic snzymes into an aggregate is still unknown. Search-i t ling for accumulation of intermediates and studying various !fractions' in the enzyme preparation (soluble versus sedi-

1 !mentable material) for the enzyme activity might provide J . !additional insight into the nature of the ser-6 mutant. I An alternative explanation for the behavior of

J ser-6 could be that in vivo the activity of one or more of I the serine biosynthetic enzymes is inhibited by some sub- i I stance which is r~moved during preparation of the enzyme I extracts& An example of this phenomenon is found in the

1 work of Suskind and Kurek who studied tryptophan - re- I quiring (td) mutants which are unable to utilize indole I for growth and which lack tryptophan synthetase activity (32). They found a mutant, td24, which requires try ptophan for growth at 25° C, but which grows sl~wly with out tryptophan above 30° C, and at this temperature also forms a slight amount of tryptophan synthetase. They were able to obtain highly active tryptophan synthetase from this mutant grown at 25° C, by suitable fractionation of crude inactive extracts. The active enzyme obtained by 54

r~-_x--~--< ...... ,--.._~---~~...... -.--~·~-- ~ ·· ·-----~·---·~-~--- ~--··- ···- -~--· ------~-~---~ --~~-·-·------~------~-- ---H-- IthiS :fractionation method was :found to differ from the

~ enzyme obtained from wild-type in its abnormally high sen sitivity to inhibition by zinc ions. Thus it seems that JI !the mutation has caused this enzyme to become sensitive to i !zinc (or some other metal inside the cell whose effect ' !could be mimiced by zinc) at 25° C. The process of fractionation in fact removed this metal from the enzyme and .i hence removed the inhibitor of' the enzyme's activity. Another possible explanation for the results ob . tained with ser-6 is that the two pathways studied by

1 Sojka and Garner may not be the major pathway of serine !biosynthesis in Neurospora crassa. The fact.that this mutant strain does not grow on glycine, while glycine and serine are thought to be interchangeable (JJ), suggests i that glycine might precede serine in the major serine bio synthetic pathway. In that case, ser-6 could be deficient in serine aldolase which is the enzyme involved in synthe- ' sis of serine from glycine (J4). Evidence that serine may be derived from glycine in ·

Neurospor~ was given by Sojka (17) and was confirmed by Maxwell using radioactive glycine (18). Abelson and Vogel

obtained evidence from labeling studies in Neurospor~ 14 ;---crassa tha t rad 1oac. t'·1v1 't y · f rom c -g1 yc1ne· 1s· 1ncorpora· t e d : into serine and cysteine (35). Pitts --et al. observed that 14 ! in rat kidney serine is synthesized from c -glycine (J6). i De Bciso et al. observed that in baker's yeast the synthe- ; :sis of serine takes place by condensation of glycine with 55

~~--~;-~:.::~;~b~;;_--;~i t deriv~d·-·f~~~-ci1i:-.::f~~m~te :-Thei-;;:i~~b-l served that radioactivity from c14-glycine is incorporated . 1

I directly into serine (15). 1 A suggested precursor of glycine is glyoxylic acid. i In baker • s yeast De Boiso et al. obs~rved that [ 1-c14 ] glyoxylate and [ 1-c14) glycolate are converted into serine and glycine. Glycolic and glyoxylic ac·id labeled with C14 1 have been shown to be effective precursors of glycine in ·! animal tissues (11, 12). Wright showed that a mutant of Neurospora crassa which requires glycine or serine also utilizes glyoxylic acid or glycolic acid. She suggested that in the biosynthesis of glycine and serine by Neuro

~ora crassa, glycolic acid can be oxidized to glyoxylic acid which can then be aminated to glycine (16). Results contradicting Wright's suggestion have been reported by another group of investigators. Combepine and Turian were unable to grow ser-1 on glyoxylate and hence concluded that this pathway must not play a major role in glycine synthesis in Neurospora (37). Maxwell determined that the difference in Wright's and Combepine's results could be

explained by the difference in carbon source used by these two investigators (18). Combepine --et al. used sucrose in their media and no serine-glycine auxotrophs utilized gly oxylate for growth. Wright used glycerol, in which case the seri.ne-glycine auxotroph was able to utilize glyoxylate,. Maxwell (18) retested mutants studied by Comb~pine and

I : Turian on glycerol and shewed that all of them utilize L~ .... 56

r·-·-·-----·-····------··-·------·------······'"-·-·---· .... -... ---...... ·------.... ·------·--1 Iglyoxylate on this carbon source. This observation indi- 1 cated that the carbon source plays an important role in I I the utilization of glyoxylate. An example of the importance of carbon source in serine and glycine metabolism is found in the work of Ulane and Ogur (38) who studied serine-glycine biosynthesis in Saccharomyces. They ob served that in this organism both the phosphorylated path : way from products of glycolysis and the glyoxylate pathway : from tricarboxylic acid cycle intermediates were operative.

! l The phosphorylated pathway appeared to be the principal biosynthetic pathway to serine and glycine during growth on glucose media, while the glyoxylate pathway was the

major pathway durin~ growth on acetate. To explain their results, they suggested that the glyoxylate pathway to glycine is repressed by growth on glucose¥ specifically, at isocitrate lyase. The repression of isocitrate lyase, which splits isocitric acid (derived from tricarboxylic acid cycle) to form succinic acid and glyoxylic acid, has been observed also in Neurospora by Sjogren and Romano (39). Flavell has observed derepression of the enzymes of the glyoxylate shunt after transfer of cultures from sucrose medium to one in which acetate was used as the carbon source (40). So it would appear that on the suc rose medium used in the present study tricarboxylic acid cycle intermediates cannot be utilized as precursors of l' glyoxylic acid. An alternative precursor for glyoxylic acid could be glycolic acid. Glycolic acid has been shown 57

rby--~~~-~-.. i~;~-~t-l-g~t~;~· -t~- --b~. --~~---~-ff;c--ti.;·~---p;;;c u~~-0-;--;:r-gi-y:l loxylic acid (11, 12, 16). Tolbert and Cohan studied syn lthesis of glycine in barley and wheat leaves. They used ' lo 14 labeled glycolic acid and found that the major prod- 1 :uctsi formed from glycolic acid are glycine and serine (1J). /Another alternative precursor of glyoxylic acid suggested ' by Hardy and Quayle is methanol which is utilized by Pseu _\domonas AM1 to synthesize glycine and serine via glyoxy-

! late (41). Whether the major pathway of serine biosynthesis ' linvolves methanol, glycolic acid, glyoxylic acid, and/or glycine is unknown. To get more information-about the pathway of serine synthesis and the location of the bio chemical lesion in ser-6 extensive studies involving metabolism of uniformly labeled methanol, glycolic acid, glyoxylic acid, glycine and serine in both ser-6 and wild type will be necessary. Another explanation for the results obtained with · ser-6 is that the mutant may not be defective in serine

bios~mthesis at all, but may be defective in the metabo lism of active "01" units. Serine has been implicated as

the reservoir for "C1" units in yeast (15) and may serve the same function in Neurospora. If the production of "01" units from other sources were blocked in ser-6,

serine might be channeled into reactions requiring "01" units, To test this possibility for ser-6, growth of the mutant could be evaluated on sodium formate or formaldehyde. 58

,----·-----·-···--·-·-·· ----···---·--·------·-· ...... ------.... ------·--·------~ I. The :fact that ser-1, ser-4, and ser-6 show no alte!'-1 ations in the biosynthetic pathways leading to serine which! have been proposed by Sojka and Garner suggests that serine i lI !metabolism in Neurospora is not yet fully understood. The i I !simplest eA~lanation for these mutants is that an altern- iative pathway for serine biosynthesis exits in Neurospora l :which remains to be elucidated. Alternatively these mu tants may be defective in their ability to form a func tional enzyme aggregate required for serine biosynthesis or they may be regulatory in nature. Clearly, additional study of serine biosynthesis in Neurospora will be neces sary to explain these paradoxical mutants. BIBLIOGRAPHY

I 1. G. A. Sojka and H. R. Garner. 1967. The serine bio-' synthetic pathway in Neurospor~ crassa. Biochim. Biophys. Acta 148, 42. 2. A. Ichihara and D. M. Greenberg. 1957. Further studies on the pathway of serine formation from carbohydrate. J. Biol. Chern. 224, 331. 3. J. E. Willis and H. J. Sallach. 1962. Evidence for a mammalian D-glyceric dehydrogenase. J. Biol. Chern. 221, 910. 4. D. A. Walsh and H. J. Sallach. 1966. Comparative studies on the pathways for serine biosynthesis in animal tissues. J. B:i.ol. Chern. 2l~1, 4068. 5. J. Hanford and D. Davies. 1958. Formation of phos phoserine from )-phosphoglycerate in higher plants. Nature 182, 532. 6. H. E. Umbarger, M. A. Umbarger, and P. M. L. Siu. 1963. Biosynthesis of serine in Escherichia coli · and Salmonela tyPhimurium. J. Bacterial • .§.2,--- 1431. 7. M. Ponce-De-Leon and L. Pizer. 1972. Serine bio synthesis and its regulation in Bacillus subtilis. J. Bacterial. 11Q, 895. 8. J. D. Nelson and H. B. Naylor. 1971. The synthesis of L-serine by Micrococcus ±xsodeikticus. Can. J. Microbial. !Zp 73. --- 9. G. J. Germano and K. E. Anderson.· 1969. Serine biosynthesis in Desulfovibrio desulfuricans. J. Bacteriol, 99, 893. -- 10. L. Pizer, M. Ponce-De-Leon, and J. Michalka. 1969. Serine biosynthesis and regulation in Haemophilus influenzae. J. £3aEteriol. 21• 1357. - 11. A. Weissbach and D. Sprinson. 1953. The metabolism· of 2-carbon compounds related to glycine. J. Biol. Chern. 203, 1023. 12, s. Weinhouse and B. Friedmann. 1951. Metabolism of labeled 2-carbon acids in the intact rat. J. Biol. Chem. 121, 707.

59 60

----·-·------···--····------··--··--···------.. ----~

N. Tolbert and M. Cohan. 1953. Products formed 1 from glycolic acid in plants. J. Biol. Chem. 204,1 649. --- l 14. G. Gilvarg and K. Bloch. 1951. The utilization of I acetic acid for amino acid synthesis in yeast. I J. Biol, Chern. !22• 339. ! 15. J. F. De Boiso and A. o. M. Stoppani. 1967, Metab i olism of serine and glycine in baker's yeast. Biochim. Biophys. Acta. 148, 48. 16. B. E. Wright. 1951. Utilization of glyoxylic acid and glycolic acid by a Neurospora mutant requir ing glycine or serine. Arch. Biochem. JiiophJI:f:i.• .J!, 332. G. A. Sojka. 1967. Ph. D. Thesis, Purdue Univer sity, Lafayette, Indiana. 18. J. B. Maxwell. 1970. Synthesis of L-amino acid oxidase by a serine or glycine-requiring strain of Neurospora. Ph. D. Thesis, California Insti- tute of Technology. Pasadena, California. 19. v. W. Woodward, J. R. DeZeeuw, and A. M. Srb. 1954. The separation and isolation of particular bio chemical mutants of Neuro~ora crassa by differ- : ential germination of conJ.dia, followed by filter ation and selective plating. Proc. Natl. Acad. Sci. u. s . .i2_, 193. --- 20. N. H. Horowitz. 19l-1-7. Methionine synthesis in }:'leurospora, The isolation of cystathionine. J. Biol. Chern. ~. 255. 21, H. J. Vogel. 1956. A convenient growth medium for Neurospora (medium N). Microbial. Genet. Bull. I.J, 42. 22. M. Westergaard and H. K. Mitchell. 1947. Neuro spora v. A synthetic medium favoring sexual re production. Am. J. Bot • .2i, 573. 23. A. G. Gornall, C. J. Bardawill, and M. M. David. 1949. Determination of serum protein by means of the Biuret reaction. J, Biol, Chern. !ZZ, 751. 24. B. N. Ames, 1966. Methods in Enzr;ology VIII (ed, s. P. Colowick and N. o. Kaplan~ Academic Press, I p. 115. I j '-~-' 62

---·------·------·---~---·-·----~------1 G. Comb~pine and, G. Turian. ~965. Recherches Sur i la b~osyntheses de la glyc~ne chez Neurospora ! crassa type sauvage et mutants. Path. Microbial. : 2~, 1o1a. . -- I J8, R. · Ulane and M. Ogur. 1972. Genetics and physiolog ic.al control of serine and glycine biosynthesis in Saccharomyces. J. Bacterial. !Q2, 34. 39. R. E. Sjogren and A. H. Romano. 1967. Evidence for multiple forms of isocitrate lyase in Neurospora crassa. J. Bacterial. 2l• 16J8. 40. R. B. Flavell. 1966. The glyoxylate cycle in Neurospora crassa. Heredity. 21, 343. 41. w. Hardy and J. R. Quayle. 1971. Aspects of gly cine and serine biosynthesis during growth of Pseudomonas AM1 on c1 compounds. Biochem. J. 121, 76J.

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