Biochemical Genetics, Vol. 16, Nos, 9/10, 1978
Genetic and Biochemical Aspects of Trehalase from
Drosophila melanogaster
Melvin J. Oliver,~ R. E. Huber, z and John H. Williamson 1, 3 Received I Feb. 1978 Final 21 Apr. 1978
Only one molecular form of trehalase (E.C. 3.2.1.28) was detectable in adult Drosophila melanogaster by polyacrylamide gel electrophoresis and isoelectric focusing. An examination of duplication- and deletion-bearing aneuploids exhibiting dosage sensitivity indicated that the enzyme is encoded by a gene, Treh +, located between 55B and 55E of the second chromosome. The tissue-specific soluble and particulate forms of trehalase appear to be manifestations of a single protein encoded by a single gene. KEY WORDS: trehalase; Drosophila; segmental aneuploidy.
INTRODUCTION The disaccharide trehalose (1 ~-D-glucopyranosyl-~-D-glucopyranoside) is the principal sugar in the hemolymph of many species of insects (Wyatt, 1967;Elbein, 1974) and functions as a source of energy for flight muscle activity. The specific hydrolase, trehalase (TX-glucoside-l-glucohydrolase, E.C. 3.2.1.28), splits trehalose into two glucose moieties, and may play an important role in the transport of glucose between tissues (Wyatt, 1967). A soluble form of trehalase is found in flight muscle, hemolymph, and the abdomen, and an "insoluble" form is associated with particulate fractions of muscle homogenates. The subcellular localization of the two forms of the This work was supported by NRC Grants A5860 to J. H. Williamson and A4691 to R. E. Huber. 1 Department of Biology, The University of Calgary, Calgary, Alberta, Canada T2N 1N4. Department of Biochemistry, The University of Calgary, Calgary, Alberta, Canada T2N 1N4. 3 Address all correspondence to J. H. W. 927
0006-2928/78/1000-0927505.00/0 © 1978PlenumPublishingCorporation
928
Oliver, Huber, and Williamson
enzyme in flight muscle has not been established. The soluble abdominal form is found in the epithelial tissue of the gut wall. There have been a number of reports suggesting that the different forms of trehalase are manifestations of different proteins. Gussin and Wyatt (1965) and Wyatt (1967) demonstrated the abdominal enzyme to be distinct from the muscle forms in Hyalophora cecropia by virtue o f p H optima and substrate affinity. Later, Friedman and Alexander (1971) and Friedman (1975) showed that in Phormia regina abdominal trehalase was electrophoretically distinct from trehalase found in flight muscle and suggested that there are two isozymes of this enzyme. Isozyme A is restricted to the midgut and blood while isozyme B is restricted to head, muscle, and rectal papillae. Similarly, Talbot and Huber (1975) showed that in Apis mellifera the two forms of the enzyme differ in their kinetic and electrophoretic properties and that in D. melanogaster there are two electrophoretic forms of trehalase. However, Marzluf (1969) concluded from heat inactivation studies that trehalase in D. melanogaster is a single enzyme, and Yanagawa (1971) reported that there were'no differences between the electrophoretic patterns of trehalases from the gut and muscle of B. mori larvae. Tenebrio molitar, Melanoplus differentialis, Leucophaea madera, and Musca domestica each exhibit a single electrophoretic species of trehalase (Talbot and Huber, 1976). Thus there is a basic uncertainty concerning the number of forms of trehalase in insects. We felt that identification of the genetic units involved in trehalase production would add significantly toward a solution to this problem. We now present the results of a combined genetic and biochemical study which indicate that D. melanogaster has only one form of trehalase encoded by one gene.
MATERIALS AND METHODS Stocks
The Y-autosome translocations used in this study were obtained from Dr. D. L. Lindsley. T (X" Y) B26 was obtained from Dr. J. R. Merriam and ysx. yL, In(1)EN, y; In(2LR) SM1 / Sco was obtained from Dr. R. C. Gethmann. An Oregon-R stock was used as a wild-type strain for comparative purposes. All of these mutants and special chromosomes are described in detail in Lindsley and Grell (1968), Lindsley et al. (1972), and Stewart and Merriam (1974). Culture Conditions
Crosses were made in eight-dram shell vials on cornmeal-yeast-sugar-agar medium (Lewis, 1960) and incubated at 25 C. Most crosses utilized two pairs
TrehalasefromD. melanogaster
929
of parents per vial and five vials. In some crosses the viability of aneuploid progeny was low and five pairs of parents per vial were used. Crosses to dissect region 54F-57B were made with two pairs of parents per vial and ten vials per genotypic comparison.
Construction of Segmental Aneuploids The basic scheme for the production of segmental aneuploids was described by Lindsley et al. (1972) and involved crossing individuals bearing Y-autosome translocations with different autosomal breakpoints and breakpoints in opposite arms of the Y-chromosome. In this scheme adjacent-I disjunction in both parents yields progeny of one sex which are deficient for the region in question while the other sex carries the reciprocal duplication. Alternate disjunction in both parents yields euploid males and females which are heterozygous for one of the two parental translocations. These euploid individuals served as controls for comparison with enzyme activities in aneuploid individuals. The protocol for screening the genome of D. melanogaster for the gene(s) for trehalase was essentially that designed by O'Brien and Gethmann (t973). In the initial screen (see Table I) only duplications were constructed and examined. In the case of regions 47E-50C and 50C-52E, translocations with breakpoints in opposite arms of the Y chromosome were not available. Attempts to examine these regions involved crosses between stocks carrying translocations with breaks in the same arm of the Y chromsome (O'Brien and Gethmann, 1973). To screen the X-chromosome for dosage-sensitive regions, hyperploids for the distal and proximal halves of the X chromosome were constructed using the X-Y translocation stock (B26) of Stewart and Merriam (1974). Fourth chromosome aneuploidy was investigated in triplo-4 flies produced by crossing y v ac females with males carrying an attached-fourth chromosome [C(4)RM/O; spaP°q. Preliminary experiments as well as earlier experiments by Marzluf (1969) revealed that trehalase activity in 24-hr-old flies is several times greater than that in freshly eclosed flies and that freezing and lyophilization did not interfere with the assay foi~trehalase activity. To minimize the effects of age within our comparisons, all experimental animals were collected within 12 hr of eclosion, aged on fresh culture medium for 12 hr, and frozen in liquid nitrogen. After freezing, the animals were lyophilized using a Virtis model 10-147 MR-BA unit and stored at - 8 0 C.
Enzyme Preparation Single-fly assays were performed in the initial screen. Regions exhibiting an
930
Oliver, Huber, and Williamson
increase in enzyme activity in the hyperploid genotypes, compared with the euploid controls, were further examined utilizing a multiple-fly preparation. For the detailed genetic dissection of regions putatively carrying structural genes for trehalase, multiple-fly preparations were used. In all determinations, triplicate assays were performed.
Single-Fly Preparation Single thoraces or abdomens were weighed and homogenized in 0.5 ml of isolation buffer (0.03 M sodium citrate,pH 5.6) in an ice bath. The homogenate was then centrifuged at 8000g for 30 min at 4 C in an Eppendorf 3200 centrifuge. A 0.2-ml aliquot of the resultant supernatant was removed for measurement of soluble trehalase activity and a further 0.2-ml aliquot was removed for use as a heat-inactivated enzyme control. In the case of the thorax preparations the remaining supernatant was discarded and the pellet was washed in 0.5 ml of isolation buffer and recentrifuged. The resultant pellet was resuspended in 0.2 ml isolation buffer and analyzed for insoluble trehalase activity.
Multiple-Fly Preparation Ten thoraces or abdomens were homogenized in 1.0 ml of isolation buffer in an ice bath. The resultant homogenate was then centrifuged at 8000g for 30 min at 4 C. Aliquots of the supernatant (0.05 ml) were removed for measurement of soluble trehalase activity and 0.2-ml samples were taken for assay of soluble protein. In the case of thorax preparations the remaining supernatant was discarded, and the pellet was resuspended, washed in 1.0 ml of isolation buffer, and then centrifuged at 8000g for 30 rain. The washed pellet was resuspended in 1.0 ml of buffer, and 0.05-ml samples were assayed for insoluble trehalase activity. For assays of insoluble protein, 0.1-ml samples were removed and mixed with 0.1 ml of 0.2 Y sodium hydroxide. The samples were placed in a boiling water bath for 10 rain prior to protein determination (Lowry et al., 1951). In all comparisons heat-treated samples were assayed for enzyme activity. Enzyme Assay
Trehalase activity was determined b y t h e method of Huber and Lefebvre (1971). Samples of extracts, reagent blanks, and glucose standards were incubated for 20 min at 30 C in 1.0 ml of 0.1 M e,~'-trehalose in 0.03 M sodium citrate buffer (pH 5.6). The reaction was terminated by placing the tubes in a boiling water bath for 4 min. One milliliter of Glucostat reagent (Worthington Biochemical Corp.) in 0.1 M phosphate buffer (pH 7.0) was introduced after cooling and the mixture was incubated for 30 min at 30 C. The reaction was
TrehalasefromD. melanogaster
931
stopped by addition of 0.1 ml of 3 N HC1. Absorbance was measured at 420 nm and compared to glucose standards. In the case of single-fly assays, enzyme activity was expressed as #moles of trehalose utilized/min/mg of dry weight. For multiple-fly assays, enzyme activity was expressed as #moles of trehalose utilized/rain/rag of protein. In all cases the background glucose was at a low level. The validity of the conditions used for the measurement of trehalase activity was tested by means of a kinetic assay. Under these conditions the activity of the enzyme was directly proportional to incubation time within the time range used for the working assay.
Analytical Disk-Gel Electrophoresis Electrophoresis was performed using the method of Ornstein and Davis (1964). Fifty flies, 50 thoraces, or 50 abdomens were homogenized in 0.2 ml of 0.03 M sodium citrate buffer (pH 5.6), and to this homogenate an equal volume of glycerol was added. The mixture was then layered onto a 7.5% polyacrylamide gel and electrophoresed for 30 rain at 5mA in tris-glycine buffer with a running pH of 9.3.
Trehalase-Specific Stain Gels were stained for trehalase activity by a modification of the procedure of Gabriel and Wang (1969) for indentification of reducing substances on polyacrylamide gels. Gels were incubated for 5 rain in 0.5 M sodium citrate buffer (pH5.6) and then incubated for 20 rain at 30 C in 0.03 M sodium citrate buffer (pH 5.6) containing 0.1 M ~,~'-trehalose. They were then washed with distilled water and immersed in 0.1 g iodoacetamide for 5 rain, rinsed again with distilled water, immersed in a 0.2% tetrazolium red solution in tubes covered in aluminum foil to exclude light, and placed in a boiling water bath for 4 rain. Developed gels were stored in 7% acetic acid.
Analytical Isoelectric Focusing Isoelectric focusing of crude extracts prepared from mass homogenates (100 whole flies, abdomens, or thoraces in 1.0 ml of 0.03 M sodium citrate buffer, pH 5.6) was performed according to the method of Vesterberg and Svensson (1966). The homogenate was spread evenly throughout a glycerol gradient in an LKB 8101 column and 500 V was applied. The pH gradient within the glycerol gradient was constructed by inserting 2.0 ml of a 40% Biolyte carrier ampholyte solution with a p H range of 4-6. Each homogenate was isoelectricfocused for 48 hr at 4 C. Fractions (1.5 ml) were collected at a rate of 1.0
932
Oliver, Huber, and Williamson
ml/min and aliquots (0.1ml) of each fraction were assayed for trehalase activity. The p H of each fraction was measured at 4 C using a combination electrode attached to a Radiometer model 26 p H meter.
RESULTS Localization of the Structural Gene for Trehalase
Table I lists the crosses used in the initial screen to produce duplications for all of the regions of the genome of D. melanogaster except 83DE on the right arm of chromosome 3. The size of the duplications varied from 100 to 215 chromomeres, with an average size of approximately 150. The cross y w f x T (X. Y) B26 produced aneuploids of both the distal and the proximal halves of the X chromosome, each duplication being much larger than the average autosomal duplication. The relative viability of aneuploids, calculated as the ratio of aneuploid flies to euploid sibs of the same sex, generally ranged from 0.1 to 1.0. In the case of regions 38C-41, 47E-50C, and 83EF-86B, no aneuploids were recoverd. Duplications for these regions have previously been recovered (O'Brien and Gethmann, 1973) and their absence in our crosses is puzzling. For regions 57B-59B and 59B-60F the relative viabilities were calculated to be 1.78 and 2.61, respectively, from total fly counts of 262 and 627. In both cases the duplications were constructed using stock T (Y;2)P59. This stock may be unusual in its segregation properties or may exhibit position effects affecting aneuploid viability. Duplication of the appropriate region of the genome containing the structural gene of an enzyme would theoretically be associated with a 50% increase in activity of that enzyme. As shown in Table I, region 54F-57B, when duplicated, was associated with an approximate 50% increase in all three major forms of trehalase. In addition, region 96A-97F was associated with an approximate 50% increase in abdominal trehalase activity. Crosses to generate these aneuploids and determinations of trehalase activities were repeated and the results are presented in Table II. Region 96A-97A was duplicated in both males and females by a reciprocal cross of stocks Jl16 and D221, since R128 was lost, and in both cases the ratios of abdominal trehalase activity in aneuploid individuals v. euploid individuals, compared on a total soluble protein basis, approximated 1.0. When the duplication was recovered in females, the ratio of activities per milliliter of extract was 1.6, which was reduced to 0.8 when calculated on a protein basis because of a 102% increase in soluble protein in aneuploid females. This phenomenon did not occur when the duplication was recovered in males, where both activity per milliliter of extract and soluble protein were approximately equal in both preparations. Apparently region 96A-97F con-
Table I, Characteristics of Trehalase Activity in Segmental Aneuploids (Duplications) of D. melanogaster Activity ratios b
Region
Parents a
Abdomen
21A-25A 25A-27E 27A-30F 30F-35BC 35BC-38C 38(;-41 40-43C 43C -45F 45F-47E 47E-50C 50C-52E 52E-54F 54F-57B 57B-59B 59 B~50F 61A-64E 64E-67C 67C-70A C 70A~74A 74A-79D 79D-83CD 83EF-86B 86B-88C 88C-91B 91B-93F 93F-96A 96A-97F 97F-100F 101A-102F 1A-9C 9C-20F
C(1)dxJ96 J96 × H52 H52xL52 L52xR15 R15 × BllO B177 x BIIO D20 x R155 R155 x L23 B107 x L23 BI07 × LllO e R14 × Ll10 e H149 × R14 L107 x H149 P59 × L107 C (1) d × P59 B141 × C( I) d B141 × G122 G122 x A31 A31 × D228 J162 × D228 L132 × J162 L136 × R36 G48 × R36 A89×G48 B93 × A89 G73 × B93 R128xG73 C(1) a x R128 y v ac × C(4) f y w f x B26 y w f × B26
0.99 0.75 1.11 1.13 0.93 0.83 0.88 0.82 -0.89 0.93 1.38 1.08 1.15 1.04 0.95 1.07 0.86 0.87 0.85 -0.85 1.13 1.10 0.75 1.46 0.91 1.22 0.92 0.77
Soluble thorax
Insoluble c thorax
0.93 1.01 1.14 1.12 0.76 -0.84 0.89 1.06 -1.05 1.12 1.45 1.21 0.86 0.95 1.00 0.95 0.81 0.98 0.99 -0.98 1.13 0.92 0.89 1.12 1.08 0.99 1.14 0.88
1.11 0.98 1.09 1.07 0.77 -0.96 0.77 1.12 -0.98 1.19 1.60 1.03 0.93 0.73 0.99 0.92 0.95 0.71 0.81 -1.04 0.88 0.88 0.98 1.19 1.01 1.01 1.19 0.80
a Unless otherwise stated, all female parents were of the genotype C ( l ) RM, y / T ( Y , 2 ) / C y or (C) 1RM, y2 bb / T(Y;3) / In (3LR) TM6, Ubx67be and the male parents were Y s x . yL, In(1) EN, y / T(Y;'2) / Cy or y s x . yL, y / T ( Y ; 3 ) / In(3LR) TM6, Ubx67be, where Cy is either In(2L + 2R) Cy, Cy cn 2 or I n ( 2 L R ) S M 1 , al z Cy cn 2 sp z. b Activities are measured as #moles of trehalose hydrolyzed m i n - 1 m g dry weight- 1. Measurements are the mean of five single-fly determinations and calculated as the ratio of activity in hyperploid to activity of euploid sibs of the same sex. c Insoluble thorax determinations are activities measured from suspensions of washed 8000g pellets of thorax homogenates. a C(1) RM, y v bb / Y. e ys X. yL, In(1)EN, y / T(Y:2) Ll10 / Sco because Y breaks in Ll10, B107, and R14 are all in yL. See Materials and Methods for explanation. f y s X. yL, y wa / 0," C ( 4 ) R M , spaP°t / O.
934
Oliver, Huber, and Williamson Table II. Trehalase Activity Measurements of Putative Dosage-Sensitive Loci Discovered in the Initial Screen (Table I) a Activity Ratios
Region
Sex
Assay
Abdomen
Soluble thorax
Insoluble thorax
54F-57B
F F F F F M
A A' A' B B B
1.11 1.21 --0.80 0.97
1,28 1.25 1.27 1.18 ---
1.61 1.38 1.26 1.32
96A -97F
a Assay A is the original single-fly assay involving five single-fly determinations. Assay A' involves ten single-fly determinations. Activities are expressed as #moles of trehalose utilized m i n - 1 m g dry weight-1. Assay B is the multiple-fly assay, and activities are expressed as #moles of trehalose utilized rain- 1 m g of protein 1. Aneuploids for region 96A-97F were constructed from reciprocal crosses of stocks D221 and Jl16, because of loss of stock R128.
tains a gene (or set of genes) that increases the total soluble protein in female abdomens when present in three doses, This phenomenon was not investigated further. Flies aneuploid for region 54F-57B had significantly greater amounts of activity of soluble and insoluble muscle trehalase activity than their euploid siblings (Table II). Single-fly assays of abdominal trehalase in flies hyperploid for this region showed a slight increase in activity. These results indicate that the structural gene(s) for soluble and insoluble muscle trehalases and possibly abdominal trehalase is situated within region 54F-57B on the right arm of the second chromosome. Region 54F-57B was genetically dissected (Fig. 1) in an attempt to obtain a more precise localization of the dosage-sensitive locus. The region was successfully subdivided into six smaller regions using crosses which allowed enzyme determinations for both hypoploid and hyperploid genotypes (Table III). These results demonstrate that there is a specific region that is associated with increased trehalase activities in all three preparations in hyperploid genotypes and with decreased activities in hypoploid genotypes. Thus the assignment of trehalase dosage sensitivity to a specific locus within the segment delineated by bands 55B and 55E of the second chromosome can be
Trehalase from D . m e l a n o g a s t e r 54
935
55
56
57
54F-55B
54F - 55E
55E-56C 55B
-56C
5 6 C - 57B
56D -57B
56E/F-57B
Fig, 1. Cytological map of region 5 4 1 : - 5 7 C on the right arm of chromosome 2. The lengths of the solid lines represent the lengths of regions duplicated and deleted by experimental crosses.
Table IIl. Trehalase Activity o f Segmental Aneuploids Within Region 5 4 F - 5 7 B a
Ratio aneuploid:euploid Region 54F-55B 54F--55E 55E-56C 55B 56C 56C-57B 56D-57B
Dose
Abdomen
Soluble thorax
Insoluble thorax
1 3 1 3 1 3 1 3
1.04 1.26 0.78 1.25 0.98 1.00 0.71 1.31
1.07 1.08 0.78 1.29 1.12 1.02 0.82 1.33
0.99 0.98 0.75 1,61 1.08 0.91 0.83 1.58
I
--
--
--
3
0.99
0.98
0.91
1.03
0.84
1
--
3
1.06
a All activities are expressed as #moles of trehalose utilized min l m g protein- 1 from multiple-fly assays. Values reported are averages of at least two determinations, and for regions 5 4 F 5 5 B , 5 5 B - 5 6 C , and 5 4 F - 5 5 E results from reciprocal crosses are included.
936
Oliver, Huber, and Williamson
made. No other region within 54F-57B exhibits such a dosage effect on trehalase activity. Electrophoretic Studies
All of the above results suggest that the three forms of trehalase in D. melanogaster are manifestations of one protein coded for by a single gene. To test this possibility homogenates of whole flies, abdomens, and thoraces were compared by polyacrylamide gel electrophoresis (Fig. 2). In each case only one band of trehalase activity was present and had a relative mobility of 0.63
Fig. 2. Electrophoreticpattern of trehalase activity in tissue preparations of D. melanogaster. W, Whole fly; T, thorax; A, abdomen. 1, Trehalase activity; 2, tracking dye, bromophenol blue; 3, low molecular weight contaminants.
Trehalase from D. melanogaster
0.10
I
i
I
o, =
Genetic and Biochemical Aspects of Trehalase from
Drosophila melanogaster
Melvin J. Oliver,~ R. E. Huber, z and John H. Williamson 1, 3 Received I Feb. 1978 Final 21 Apr. 1978
Only one molecular form of trehalase (E.C. 3.2.1.28) was detectable in adult Drosophila melanogaster by polyacrylamide gel electrophoresis and isoelectric focusing. An examination of duplication- and deletion-bearing aneuploids exhibiting dosage sensitivity indicated that the enzyme is encoded by a gene, Treh +, located between 55B and 55E of the second chromosome. The tissue-specific soluble and particulate forms of trehalase appear to be manifestations of a single protein encoded by a single gene. KEY WORDS: trehalase; Drosophila; segmental aneuploidy.
INTRODUCTION The disaccharide trehalose (1 ~-D-glucopyranosyl-~-D-glucopyranoside) is the principal sugar in the hemolymph of many species of insects (Wyatt, 1967;Elbein, 1974) and functions as a source of energy for flight muscle activity. The specific hydrolase, trehalase (TX-glucoside-l-glucohydrolase, E.C. 3.2.1.28), splits trehalose into two glucose moieties, and may play an important role in the transport of glucose between tissues (Wyatt, 1967). A soluble form of trehalase is found in flight muscle, hemolymph, and the abdomen, and an "insoluble" form is associated with particulate fractions of muscle homogenates. The subcellular localization of the two forms of the This work was supported by NRC Grants A5860 to J. H. Williamson and A4691 to R. E. Huber. 1 Department of Biology, The University of Calgary, Calgary, Alberta, Canada T2N 1N4. Department of Biochemistry, The University of Calgary, Calgary, Alberta, Canada T2N 1N4. 3 Address all correspondence to J. H. W. 927
0006-2928/78/1000-0927505.00/0 © 1978PlenumPublishingCorporation
928
Oliver, Huber, and Williamson
enzyme in flight muscle has not been established. The soluble abdominal form is found in the epithelial tissue of the gut wall. There have been a number of reports suggesting that the different forms of trehalase are manifestations of different proteins. Gussin and Wyatt (1965) and Wyatt (1967) demonstrated the abdominal enzyme to be distinct from the muscle forms in Hyalophora cecropia by virtue o f p H optima and substrate affinity. Later, Friedman and Alexander (1971) and Friedman (1975) showed that in Phormia regina abdominal trehalase was electrophoretically distinct from trehalase found in flight muscle and suggested that there are two isozymes of this enzyme. Isozyme A is restricted to the midgut and blood while isozyme B is restricted to head, muscle, and rectal papillae. Similarly, Talbot and Huber (1975) showed that in Apis mellifera the two forms of the enzyme differ in their kinetic and electrophoretic properties and that in D. melanogaster there are two electrophoretic forms of trehalase. However, Marzluf (1969) concluded from heat inactivation studies that trehalase in D. melanogaster is a single enzyme, and Yanagawa (1971) reported that there were'no differences between the electrophoretic patterns of trehalases from the gut and muscle of B. mori larvae. Tenebrio molitar, Melanoplus differentialis, Leucophaea madera, and Musca domestica each exhibit a single electrophoretic species of trehalase (Talbot and Huber, 1976). Thus there is a basic uncertainty concerning the number of forms of trehalase in insects. We felt that identification of the genetic units involved in trehalase production would add significantly toward a solution to this problem. We now present the results of a combined genetic and biochemical study which indicate that D. melanogaster has only one form of trehalase encoded by one gene.
MATERIALS AND METHODS Stocks
The Y-autosome translocations used in this study were obtained from Dr. D. L. Lindsley. T (X" Y) B26 was obtained from Dr. J. R. Merriam and ysx. yL, In(1)EN, y; In(2LR) SM1 / Sco was obtained from Dr. R. C. Gethmann. An Oregon-R stock was used as a wild-type strain for comparative purposes. All of these mutants and special chromosomes are described in detail in Lindsley and Grell (1968), Lindsley et al. (1972), and Stewart and Merriam (1974). Culture Conditions
Crosses were made in eight-dram shell vials on cornmeal-yeast-sugar-agar medium (Lewis, 1960) and incubated at 25 C. Most crosses utilized two pairs
TrehalasefromD. melanogaster
929
of parents per vial and five vials. In some crosses the viability of aneuploid progeny was low and five pairs of parents per vial were used. Crosses to dissect region 54F-57B were made with two pairs of parents per vial and ten vials per genotypic comparison.
Construction of Segmental Aneuploids The basic scheme for the production of segmental aneuploids was described by Lindsley et al. (1972) and involved crossing individuals bearing Y-autosome translocations with different autosomal breakpoints and breakpoints in opposite arms of the Y-chromosome. In this scheme adjacent-I disjunction in both parents yields progeny of one sex which are deficient for the region in question while the other sex carries the reciprocal duplication. Alternate disjunction in both parents yields euploid males and females which are heterozygous for one of the two parental translocations. These euploid individuals served as controls for comparison with enzyme activities in aneuploid individuals. The protocol for screening the genome of D. melanogaster for the gene(s) for trehalase was essentially that designed by O'Brien and Gethmann (t973). In the initial screen (see Table I) only duplications were constructed and examined. In the case of regions 47E-50C and 50C-52E, translocations with breakpoints in opposite arms of the Y chromosome were not available. Attempts to examine these regions involved crosses between stocks carrying translocations with breaks in the same arm of the Y chromsome (O'Brien and Gethmann, 1973). To screen the X-chromosome for dosage-sensitive regions, hyperploids for the distal and proximal halves of the X chromosome were constructed using the X-Y translocation stock (B26) of Stewart and Merriam (1974). Fourth chromosome aneuploidy was investigated in triplo-4 flies produced by crossing y v ac females with males carrying an attached-fourth chromosome [C(4)RM/O; spaP°q. Preliminary experiments as well as earlier experiments by Marzluf (1969) revealed that trehalase activity in 24-hr-old flies is several times greater than that in freshly eclosed flies and that freezing and lyophilization did not interfere with the assay foi~trehalase activity. To minimize the effects of age within our comparisons, all experimental animals were collected within 12 hr of eclosion, aged on fresh culture medium for 12 hr, and frozen in liquid nitrogen. After freezing, the animals were lyophilized using a Virtis model 10-147 MR-BA unit and stored at - 8 0 C.
Enzyme Preparation Single-fly assays were performed in the initial screen. Regions exhibiting an
930
Oliver, Huber, and Williamson
increase in enzyme activity in the hyperploid genotypes, compared with the euploid controls, were further examined utilizing a multiple-fly preparation. For the detailed genetic dissection of regions putatively carrying structural genes for trehalase, multiple-fly preparations were used. In all determinations, triplicate assays were performed.
Single-Fly Preparation Single thoraces or abdomens were weighed and homogenized in 0.5 ml of isolation buffer (0.03 M sodium citrate,pH 5.6) in an ice bath. The homogenate was then centrifuged at 8000g for 30 min at 4 C in an Eppendorf 3200 centrifuge. A 0.2-ml aliquot of the resultant supernatant was removed for measurement of soluble trehalase activity and a further 0.2-ml aliquot was removed for use as a heat-inactivated enzyme control. In the case of the thorax preparations the remaining supernatant was discarded and the pellet was washed in 0.5 ml of isolation buffer and recentrifuged. The resultant pellet was resuspended in 0.2 ml isolation buffer and analyzed for insoluble trehalase activity.
Multiple-Fly Preparation Ten thoraces or abdomens were homogenized in 1.0 ml of isolation buffer in an ice bath. The resultant homogenate was then centrifuged at 8000g for 30 min at 4 C. Aliquots of the supernatant (0.05 ml) were removed for measurement of soluble trehalase activity and 0.2-ml samples were taken for assay of soluble protein. In the case of thorax preparations the remaining supernatant was discarded, and the pellet was resuspended, washed in 1.0 ml of isolation buffer, and then centrifuged at 8000g for 30 rain. The washed pellet was resuspended in 1.0 ml of buffer, and 0.05-ml samples were assayed for insoluble trehalase activity. For assays of insoluble protein, 0.1-ml samples were removed and mixed with 0.1 ml of 0.2 Y sodium hydroxide. The samples were placed in a boiling water bath for 10 rain prior to protein determination (Lowry et al., 1951). In all comparisons heat-treated samples were assayed for enzyme activity. Enzyme Assay
Trehalase activity was determined b y t h e method of Huber and Lefebvre (1971). Samples of extracts, reagent blanks, and glucose standards were incubated for 20 min at 30 C in 1.0 ml of 0.1 M e,~'-trehalose in 0.03 M sodium citrate buffer (pH 5.6). The reaction was terminated by placing the tubes in a boiling water bath for 4 min. One milliliter of Glucostat reagent (Worthington Biochemical Corp.) in 0.1 M phosphate buffer (pH 7.0) was introduced after cooling and the mixture was incubated for 30 min at 30 C. The reaction was
TrehalasefromD. melanogaster
931
stopped by addition of 0.1 ml of 3 N HC1. Absorbance was measured at 420 nm and compared to glucose standards. In the case of single-fly assays, enzyme activity was expressed as #moles of trehalose utilized/min/mg of dry weight. For multiple-fly assays, enzyme activity was expressed as #moles of trehalose utilized/rain/rag of protein. In all cases the background glucose was at a low level. The validity of the conditions used for the measurement of trehalase activity was tested by means of a kinetic assay. Under these conditions the activity of the enzyme was directly proportional to incubation time within the time range used for the working assay.
Analytical Disk-Gel Electrophoresis Electrophoresis was performed using the method of Ornstein and Davis (1964). Fifty flies, 50 thoraces, or 50 abdomens were homogenized in 0.2 ml of 0.03 M sodium citrate buffer (pH 5.6), and to this homogenate an equal volume of glycerol was added. The mixture was then layered onto a 7.5% polyacrylamide gel and electrophoresed for 30 rain at 5mA in tris-glycine buffer with a running pH of 9.3.
Trehalase-Specific Stain Gels were stained for trehalase activity by a modification of the procedure of Gabriel and Wang (1969) for indentification of reducing substances on polyacrylamide gels. Gels were incubated for 5 rain in 0.5 M sodium citrate buffer (pH5.6) and then incubated for 20 rain at 30 C in 0.03 M sodium citrate buffer (pH 5.6) containing 0.1 M ~,~'-trehalose. They were then washed with distilled water and immersed in 0.1 g iodoacetamide for 5 rain, rinsed again with distilled water, immersed in a 0.2% tetrazolium red solution in tubes covered in aluminum foil to exclude light, and placed in a boiling water bath for 4 rain. Developed gels were stored in 7% acetic acid.
Analytical Isoelectric Focusing Isoelectric focusing of crude extracts prepared from mass homogenates (100 whole flies, abdomens, or thoraces in 1.0 ml of 0.03 M sodium citrate buffer, pH 5.6) was performed according to the method of Vesterberg and Svensson (1966). The homogenate was spread evenly throughout a glycerol gradient in an LKB 8101 column and 500 V was applied. The pH gradient within the glycerol gradient was constructed by inserting 2.0 ml of a 40% Biolyte carrier ampholyte solution with a p H range of 4-6. Each homogenate was isoelectricfocused for 48 hr at 4 C. Fractions (1.5 ml) were collected at a rate of 1.0
932
Oliver, Huber, and Williamson
ml/min and aliquots (0.1ml) of each fraction were assayed for trehalase activity. The p H of each fraction was measured at 4 C using a combination electrode attached to a Radiometer model 26 p H meter.
RESULTS Localization of the Structural Gene for Trehalase
Table I lists the crosses used in the initial screen to produce duplications for all of the regions of the genome of D. melanogaster except 83DE on the right arm of chromosome 3. The size of the duplications varied from 100 to 215 chromomeres, with an average size of approximately 150. The cross y w f x T (X. Y) B26 produced aneuploids of both the distal and the proximal halves of the X chromosome, each duplication being much larger than the average autosomal duplication. The relative viability of aneuploids, calculated as the ratio of aneuploid flies to euploid sibs of the same sex, generally ranged from 0.1 to 1.0. In the case of regions 38C-41, 47E-50C, and 83EF-86B, no aneuploids were recoverd. Duplications for these regions have previously been recovered (O'Brien and Gethmann, 1973) and their absence in our crosses is puzzling. For regions 57B-59B and 59B-60F the relative viabilities were calculated to be 1.78 and 2.61, respectively, from total fly counts of 262 and 627. In both cases the duplications were constructed using stock T (Y;2)P59. This stock may be unusual in its segregation properties or may exhibit position effects affecting aneuploid viability. Duplication of the appropriate region of the genome containing the structural gene of an enzyme would theoretically be associated with a 50% increase in activity of that enzyme. As shown in Table I, region 54F-57B, when duplicated, was associated with an approximate 50% increase in all three major forms of trehalase. In addition, region 96A-97F was associated with an approximate 50% increase in abdominal trehalase activity. Crosses to generate these aneuploids and determinations of trehalase activities were repeated and the results are presented in Table II. Region 96A-97A was duplicated in both males and females by a reciprocal cross of stocks Jl16 and D221, since R128 was lost, and in both cases the ratios of abdominal trehalase activity in aneuploid individuals v. euploid individuals, compared on a total soluble protein basis, approximated 1.0. When the duplication was recovered in females, the ratio of activities per milliliter of extract was 1.6, which was reduced to 0.8 when calculated on a protein basis because of a 102% increase in soluble protein in aneuploid females. This phenomenon did not occur when the duplication was recovered in males, where both activity per milliliter of extract and soluble protein were approximately equal in both preparations. Apparently region 96A-97F con-
Table I, Characteristics of Trehalase Activity in Segmental Aneuploids (Duplications) of D. melanogaster Activity ratios b
Region
Parents a
Abdomen
21A-25A 25A-27E 27A-30F 30F-35BC 35BC-38C 38(;-41 40-43C 43C -45F 45F-47E 47E-50C 50C-52E 52E-54F 54F-57B 57B-59B 59 B~50F 61A-64E 64E-67C 67C-70A C 70A~74A 74A-79D 79D-83CD 83EF-86B 86B-88C 88C-91B 91B-93F 93F-96A 96A-97F 97F-100F 101A-102F 1A-9C 9C-20F
C(1)dxJ96 J96 × H52 H52xL52 L52xR15 R15 × BllO B177 x BIIO D20 x R155 R155 x L23 B107 x L23 BI07 × LllO e R14 × Ll10 e H149 × R14 L107 x H149 P59 × L107 C (1) d × P59 B141 × C( I) d B141 × G122 G122 x A31 A31 × D228 J162 × D228 L132 × J162 L136 × R36 G48 × R36 A89×G48 B93 × A89 G73 × B93 R128xG73 C(1) a x R128 y v ac × C(4) f y w f x B26 y w f × B26
0.99 0.75 1.11 1.13 0.93 0.83 0.88 0.82 -0.89 0.93 1.38 1.08 1.15 1.04 0.95 1.07 0.86 0.87 0.85 -0.85 1.13 1.10 0.75 1.46 0.91 1.22 0.92 0.77
Soluble thorax
Insoluble c thorax
0.93 1.01 1.14 1.12 0.76 -0.84 0.89 1.06 -1.05 1.12 1.45 1.21 0.86 0.95 1.00 0.95 0.81 0.98 0.99 -0.98 1.13 0.92 0.89 1.12 1.08 0.99 1.14 0.88
1.11 0.98 1.09 1.07 0.77 -0.96 0.77 1.12 -0.98 1.19 1.60 1.03 0.93 0.73 0.99 0.92 0.95 0.71 0.81 -1.04 0.88 0.88 0.98 1.19 1.01 1.01 1.19 0.80
a Unless otherwise stated, all female parents were of the genotype C ( l ) RM, y / T ( Y , 2 ) / C y or (C) 1RM, y2 bb / T(Y;3) / In (3LR) TM6, Ubx67be and the male parents were Y s x . yL, In(1) EN, y / T(Y;'2) / Cy or y s x . yL, y / T ( Y ; 3 ) / In(3LR) TM6, Ubx67be, where Cy is either In(2L + 2R) Cy, Cy cn 2 or I n ( 2 L R ) S M 1 , al z Cy cn 2 sp z. b Activities are measured as #moles of trehalose hydrolyzed m i n - 1 m g dry weight- 1. Measurements are the mean of five single-fly determinations and calculated as the ratio of activity in hyperploid to activity of euploid sibs of the same sex. c Insoluble thorax determinations are activities measured from suspensions of washed 8000g pellets of thorax homogenates. a C(1) RM, y v bb / Y. e ys X. yL, In(1)EN, y / T(Y:2) Ll10 / Sco because Y breaks in Ll10, B107, and R14 are all in yL. See Materials and Methods for explanation. f y s X. yL, y wa / 0," C ( 4 ) R M , spaP°t / O.
934
Oliver, Huber, and Williamson Table II. Trehalase Activity Measurements of Putative Dosage-Sensitive Loci Discovered in the Initial Screen (Table I) a Activity Ratios
Region
Sex
Assay
Abdomen
Soluble thorax
Insoluble thorax
54F-57B
F F F F F M
A A' A' B B B
1.11 1.21 --0.80 0.97
1,28 1.25 1.27 1.18 ---
1.61 1.38 1.26 1.32
96A -97F
a Assay A is the original single-fly assay involving five single-fly determinations. Assay A' involves ten single-fly determinations. Activities are expressed as #moles of trehalose utilized m i n - 1 m g dry weight-1. Assay B is the multiple-fly assay, and activities are expressed as #moles of trehalose utilized rain- 1 m g of protein 1. Aneuploids for region 96A-97F were constructed from reciprocal crosses of stocks D221 and Jl16, because of loss of stock R128.
tains a gene (or set of genes) that increases the total soluble protein in female abdomens when present in three doses, This phenomenon was not investigated further. Flies aneuploid for region 54F-57B had significantly greater amounts of activity of soluble and insoluble muscle trehalase activity than their euploid siblings (Table II). Single-fly assays of abdominal trehalase in flies hyperploid for this region showed a slight increase in activity. These results indicate that the structural gene(s) for soluble and insoluble muscle trehalases and possibly abdominal trehalase is situated within region 54F-57B on the right arm of the second chromosome. Region 54F-57B was genetically dissected (Fig. 1) in an attempt to obtain a more precise localization of the dosage-sensitive locus. The region was successfully subdivided into six smaller regions using crosses which allowed enzyme determinations for both hypoploid and hyperploid genotypes (Table III). These results demonstrate that there is a specific region that is associated with increased trehalase activities in all three preparations in hyperploid genotypes and with decreased activities in hypoploid genotypes. Thus the assignment of trehalase dosage sensitivity to a specific locus within the segment delineated by bands 55B and 55E of the second chromosome can be
Trehalase from D . m e l a n o g a s t e r 54
935
55
56
57
54F-55B
54F - 55E
55E-56C 55B
-56C
5 6 C - 57B
56D -57B
56E/F-57B
Fig, 1. Cytological map of region 5 4 1 : - 5 7 C on the right arm of chromosome 2. The lengths of the solid lines represent the lengths of regions duplicated and deleted by experimental crosses.
Table IIl. Trehalase Activity o f Segmental Aneuploids Within Region 5 4 F - 5 7 B a
Ratio aneuploid:euploid Region 54F-55B 54F--55E 55E-56C 55B 56C 56C-57B 56D-57B
Dose
Abdomen
Soluble thorax
Insoluble thorax
1 3 1 3 1 3 1 3
1.04 1.26 0.78 1.25 0.98 1.00 0.71 1.31
1.07 1.08 0.78 1.29 1.12 1.02 0.82 1.33
0.99 0.98 0.75 1,61 1.08 0.91 0.83 1.58
I
--
--
--
3
0.99
0.98
0.91
1.03
0.84
1
--
3
1.06
a All activities are expressed as #moles of trehalose utilized min l m g protein- 1 from multiple-fly assays. Values reported are averages of at least two determinations, and for regions 5 4 F 5 5 B , 5 5 B - 5 6 C , and 5 4 F - 5 5 E results from reciprocal crosses are included.
936
Oliver, Huber, and Williamson
made. No other region within 54F-57B exhibits such a dosage effect on trehalase activity. Electrophoretic Studies
All of the above results suggest that the three forms of trehalase in D. melanogaster are manifestations of one protein coded for by a single gene. To test this possibility homogenates of whole flies, abdomens, and thoraces were compared by polyacrylamide gel electrophoresis (Fig. 2). In each case only one band of trehalase activity was present and had a relative mobility of 0.63
Fig. 2. Electrophoreticpattern of trehalase activity in tissue preparations of D. melanogaster. W, Whole fly; T, thorax; A, abdomen. 1, Trehalase activity; 2, tracking dye, bromophenol blue; 3, low molecular weight contaminants.
Trehalase from D. melanogaster
0.10
I
i
I
o, =
