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Developmental changes in the expression of S-acyl fatty acid synthase thioesterase gene and lipid composition in the uropygial gland of mallard ducks (Anas platyrhynchos)
ARCHIVES
OF BIOCHEMISTRY
Vol. 284, No. 1, January,
AND
BIOPHYSICS
pp. 201-206,199l
Developmental Changes in the Expression of S-Acyl Fatty Acid Synthase Thioesterase Gene and Lipid Composition in the Uropygial Gland of Mallard Ducks (Anas platyrhynchos)’ P. E. Kolattukudy,’
Stewart
Ohio State Biotechnology
Center, The Ohio State University,
Received
July
18, 1990, and in revised
Bohnet,
form
Glenn
September
Sasaki,
and Linda Columbus,
14, 1990
Developmental changes in the composition of the uropygial gland secretory lipids of the postembryonic mallard ducks (Anasplatyrhynchos) were determined. During the first 3 weeks after hatching, the composition of the secretory lipids remained constant; the lipids consisted of long-chain wax esters composed of a complex mixture of n-, monomethyl, and dimethyl fatty acids esterified to n-Cl6 and n-Cl8 fatty alcohols. Afterward, as the ducks began to acquire adult feathers, short-chain wax esters composed of 2- and 4-monomethyl fatty acids began to appear with 2-methylhexanoyl and 4-methylhexanoyl as the major acyl components; esters of shortchain monomethyl fatty acids (
‘This work was supported, in part, by Grant GM-18278 from the National Institutes of Health. ’ To whom correspondence should be addressed at Ohio State Biotechnology Center, 206 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210. 0003.9861/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Rogers
Ohio 43210
Sebaceous glands produce unique lipids that serve a variety of functions on the animal surface (1,2). In birds a single large gland, called the uropygial gland, performs much the same functions as those of the numerous diminutive sebaceousglands that are widely distributed on the surface of mammals. The avian glands that are structurally and functionally analogous to the mammalian sebaceous glands present a convenient model for biochemical and molecular biological studies on this holocrine system (3-6). The synthesis of the unique lipids in the uropygial gland involves some unique proteins that function in conjunction with the usual enzymes. For example, in the goose uropygial gland, a unique cytoplasmic malonyl-CoA decarboxylase causes the production of multiple methyl branched fatty acid by removing malonyl-CoA to ensure that methylmalonyl-CoA is the only substrate available to the fatty acid synthase that synthesizes the branched acids when only the branched precursor is available (68). In the mallard duck uropygial gland, a unique 30-kDa thioesterase hydrolytically releasesshort-chain fatty acids from fatty acid synthase and thus causes production of short-chain wax esters (9, 10). Initiation of the synthesis of these tissue-specific proteins is a biochemical manifestation of the differentiation of the gland. Expression of the genes that code for such unique proteins would provide a useful parameter for studying the developmental biology of such sebaceous glands. In developing embryonic goose uropygial glands, malonyl-CoA decarboxylase transcripts appeared several days prior to hatching and reached maximal levels by hatching (11). In the developing duck embryonic uropygial glands, malic enzyme and fatty acid synthase transcripts increased dramatically several days before hatching (12). High levels of these enzymes are required for the production of the gland lipids, although these enzymes are not unique to the glands. The developmental pattern of 201
202
KOLATTUKUDY
ET
AL.
expression of the tissue-specific thioesterase gene involved in the production of the unique short-chain lipids of the gland has not been studied. In this paper we report that the expression of this gene occurs only when the mallards acquire adult feathers, replacing their down feathers. The composition of the uropygial gland lipids also reflects this change in gene expression by the replacement of longchain wax esters by the short-chain wax esters that usually are considered characteristic of mallards. MATERIALS
AND
METHODS
Materials. Ducklings were purchased from Lind’s Hatchery in Umatilla, Oregon, or from Whistling Wings in Hanover, Illinois. They were maintained in outdoor pens on standard chick mash (minus antibiotics). Dithioerythritol (DTE)3 and N,O-(trimethylsilyl)acetamide were purchased from Sigma. [iz51] Protein A (80-90 Ci/g) was from New England Nuclear. BFB (14%) in butanol was prepared by slowly bubbling BFB gas into freshly distilled n-butanol until 14% weight gain was obtained. All restriction enzymes were purchased from New England Biolabs. Nitrocellulose and Nytran membranes were from Schleicher & Schuell. Young birds (2, 7, Tissue preparations and extraction of products. and 14 days) were sacrificed by exsanguination and the uropygial glands were carefully dissected from the surrounding fat and connective tissue (3). Glands were divided into two portions: one was quickly frozen in liquid nitrogen and stored at -80°C for RNA and DNA analysis and the other was homogenized in chloroform:methanol (2:l). The lipids were recovered in chloroform according to the method of Bligh and Dyer (13) and the solvent was evaporated with a gentle stream of nitrogen. With older ducklings (3 weeks or more) the waxes were collected by gently squeezing the gland to express the lipids into a test tube where they were stored at -2O’C until further analysis. At Days 23, 31, 39, 51, and 65, two birds were sacrificed and portions of each gland were used for protein characterization and RNA and DNA extraction. Intact wax was injected into a HewlettAnalysis of the into& wan. Packard Model 5840A gas chromatograph equipped with a 10 m X 0.2 mm OV-1 capillary column and attached to a HP5985 mass spectrometer to obtain electron impact mass spectra of the intact waxes. Column temperature was set at 15O’C for 1 min, followed by a 150-280°C temperature program at lO”C/min. Mass spectra were recorded with 70 eV ionizing voltage. Wax esters were identified by their diagnostic ions representing the fatty acid portion (RCOOH:) (4). The ions representing these fragments were quantitated and tabulated as relative percentages of the total under the selected ion display (SID) option of the mass spectrometer. This option allows for the simultaneous display and quantitation of selected ion profiles derived from the total ion data. All values were standardized by comparison to the values obtained with equimolar amounts of octadecyl heptanoate and octadecyl octadecanoate. Diester waxes were identified by using ions at m/e 143, 171, and 199, corresponding to the fragments of 3-hydroxy octanoate, -decanoate, and -dodecanoate, respectively. The values obtained as relative percentages for these fragments using the SID program were identical to the values obtained following analysis of the products of transesterification of the diesters with BFB in methanol. These ions were therefore used to identify and quantitate the intact diester waxes. Compositional analysis of the wax esters. Waxes were transesterified by refluxing with 14% BF, in butanol (14). The reaction products were plated on l-mm-thick silica gel G TLC plates and developed in hexane: ethyl ether:formic acid (9O:lO:l v/v). The butyl esters were eluted from the silica gel with chloroform:methanol (2:l) and analyzed by gas chromatography on an OV-1 column (10 m X 0.2 mm). The column was maintained at 150°C for 1 min. followed by a temperature program to
3 Abbreviations
used: DTE,
dithioerythritol;
SID, selected
ion display.
I
5
15 TIME C&d)
FIG. 1.
Gas-liquid chromatograms pygial glands of mallard ducklings. 65.day-old ducklings, respectively. scribed in the text.
of intact wax esters from the uroA, B, and C represent 23-, 39-, and Capillary GLC was performed as de-
280°C at lO”/min. The alcohol was recovered from the silica gel by elution with diethyl ether and incubated with bis-N,O-(trimethylsilyl)acetamide at 90°C for 30 min to convert the alcohol to the trimethysilyl ether. These derivatives were analyzed by GLC-MS using the same column and temperature conditions as those used for the analysis of the butyl esters. Uropygial glands were dissected from Enzyme level measurements. ducklings sacrificed at 23,31,39,51, and 65 days of age, quickly frozen in liquid nitrogen, and stored at -80°C for further analysis. For protein analysis, each sample was homogenized in 0.1 M sodium phosphate buffer, pH 7, containing 0.25 M sucrose and 1 mM DTE (3 ml buffer/g tissue). Homogenates were sequentially centrifuged 5 min at lOOOg, 15 min at 27,000g and 90 min at 105,OOOg; all pellets were discarded. Total protein in the 105,OOOg supernatant was measured by the method of Lowry et al. (15). Fatty acid synthase and S-acyl fatty acid synthase thioesterase in the soluble protein were quantitated by a [lz51] protein A-immunoblot method (16). Rabbit antibody against the synthase was prepared as previously described (17) and the antibodies against the thioesterase were prepared similarly using the purified duck thioesterase (10). SDSpolyacrylamide gel electrophoresis was performed following the method of Laemmli (18), using 10% resolving and 3% stacking gels. Isolation of RNA and genomic DNA. Portions (2 g) of uropygial glands and liver were ground into powder in liquid NP, and DNA and RNA were isolated from uropygial glands by the method of Zehner et al. (19) with the addition of ultracentrifugation through CsCl gradients. After the phenol extraction and ethanol precipitation the nucleic acids
REGULATION
OF
THIOESTERASE
were dissolved in 8 ml of 1 M CsCl containing 1% P-mercaptoethanol. The solution was layered over 5 ml of 5.7 M CsCl containing 1% 2mercaptoethanol and spun at 22,000 rpm overnight in a Beckman SW28 rotor. The DNA layer (detected by its Schlieren pattern) was removed by syringe and the RNA pellet was dissolved in 10 mM Tris-HCl, pH 7.6, containing 1% 2-mercaptoethanol and 1 mM EDTA and was then ethanol precipitated. The DNA layer was mixed with CsCl to a final solution density of 1.42; 100 ~1 of 10 mg/ml ethidium bromide was added and the solution was centrifuged at 55,000 rpm overnight in a Beckman Ti70 rotor. The DNA layer was removed by syringe, butanol extracted, and dialyzed against 10 mM Tris-HCl, pH 7.6, containing 1 mM EDTA. RNA was dissolved in diethylpyrocarbonate-treated H,O. Northern blot analysis. Electrophoresis of 25 Kg RNA from each sample was run on a 1.2% agarose-formaldehyde gel overnight at 25 V (20) with RNA standards from BRL Life Technologies, Inc. The RNA was blotted onto Nytran via capillary action using 10 mM NaOH as the transfer medium (21). The blot was baked for 2 h at 80°C and hybridized with 3zP nick translated (22) S-acyl fatty acid synthase thioesterase cDNA (23) in formamide/Denhardt’s-based solution at 42’C (24).
RESULTS
Short-chain wax esters constitute the bulk of secretory lipids produced by adult mallards (25). However, gas-liquid chromatograms of intact lipids collected from ducklings of ages 2 to 21 days showed a pattern quite different from that of the normal adult. Long-chain wax esters were the major components in the ducklings (Fig. 1A). After 3 weeks of age, the GLC pattern from the intact waxes gradually changed to include components with shorter retention times (Fig. 1B). As the adult feather patterns emerged, the uropygial gland products attained a pattern identical to that of the mature mallard ducks (Fig. 1C). Diesters of 3-hydroxy fatty acids were minor components and at no time did they constitute greater than 10% of the total lipids. Analysis of the components of the diesters showed that the composition was quite similar to that previously reported (26). To determine fatty acid composition, the intact waxes from the uropygial glands of ducklings were transesterified with BFB in butanol and the butyl esters were analyzed by gas-liquid chromatography and mass spectrometry. The waxes of the hatched ducklings contained a very complex mixture of fatty acids (Table I). The pattern remained constant until the ducklings were 23 days old. The fatty acids from the glands of ducklings of 1 to 23 days of age contained a high proportion of multimethylbranched fatty acids and were longer than those found in adults. As the ducklings became older the fatty acids produced by the gland became progressively shorter and the content of multimethyl-branched acids decreased. The fatty acid composition of the wax from the 31-day-old duckling showed a transition to the more adult-like composition reached by 65 days of age, as shown in Table I. At 2 months, when adult feathers became the dominant covering of the birds, the characteristic monomethylbranched Cs acids produced by the adult mallards became a dominant component of wax esters. The most striking change in the fatty acid composition was that short-chain acids, characteristic of the adult mallard, appeared as the dominant components only at
TN
UROPYGIAL
GLAND
IN
203
DUCK
TABLE
I
Developmental Changesin the Composition of the Acyl Portion of the Uropygial Gland Wax of Ducklings % Composition Fatty
acid
Age in days:
2-Me-C, 4-Me-C, 2-Me-C!? 4-Me-C, 2-Me-C, 4-Me-Cs 2,6-Me.& 2-Me-& 4-Me-C, 2-Me-Cl0 4-Me-C0 2,6-MeZ-C,0 n-Cl1 2-Me-C1 2,6-Me&,, 4-Me-C,, 2-Me-t& 4-Me-f& 2,6-Me&, n-C14 4,8-Me&& 2-Me-f& 4-Me-Cl3 2,6,8-Me&i3 2,8-Me&s 2,6,10-Me&i3 n-C15 4,6-Me&i3 2-Me-C,, 4-Me-‘& 2,6-Me&, 4,6-Me&i4 4,8-Me&, 4,10-MeZ-C11 4-Me-& 2,6,8-Me&, 4,12-Me&,, 2-Me-& 4-Me-C& 4,6-Me&, 2,10-Me2-Cn 2-Me-Cl6 n-C16 2-Me-C,, 4-Me-Cl7 2,6-Me&s 4,6-Me&& 2,6-Me&i7 48Me&s 4,10-Me&i6 4,12-Me&,, 28Me&r 4,8-Me&i7 4-Me-C,s 4-Me-C& Total
23
31
65
0.13 0.22
16.45 24.86 3.29
0.20 0.06 0.30
0.36 2.83
3.50 8.31 10.10
1.70 3.88 0.32 0.23
6.03 6.33 0.58 0.61
1.14 0.20 0.33 1.41 1.87 1.66
1.01 3.09
8.15 9.27
6.37 4.53
1.24 1.38
1.36 0.37 1.98
1.65 0.83 1.27 1.24 5.65 2.65 1.30 2.11 2.70 2.68 1.76
2.27 2.38 1.37
0.91
1.63 5.48 7.54 1.83 3.02
3.28 3.53
1.92
1.87 1.27 4.48 1.37 2.43 0.89
2.217 2.49
2.18
2.84
0.80 5.12
2.18 2.00
5.79
2.06 2.02 2.53 3.79
3.14 3.20 1.89
2.24 1.41 0.96 1.04
0.97
0.68 2-Methyl 4-Methyl
18.37 11.89
25.75 34.78
41.58 52.58
204
KOLATTUKUDY
ET
AL. TABLE
II
The Alcohol Portion of the Wax Esters from Uropygial Glands of Developing Mallard Ducklings 5% Composition Chain length
E t i!
0
59
Day from Hatch FIG. 2. Changes in the composition of the acyl portion of the wax esters from the uropygial glands of developing mallard ducklings. Shortchain fatty acids are defined as Cl2 or less. Fatty-acid chain length was determined by GLC-MS analysis as described in the text.
2 months of age. Until after 3 weeks of age, only about 10% of the fatty acids were of short-chain length (
Age in days:
Cl6 Cl7 CL3 Cl9 C 20
23
31
39
65
0.8 22.1 55.4 11.0 10.0
1.9 27.6 47.2 13.6 9.8
2.0 43.5 38.5 10.9 5.2
4.2 36.3 31.1 12.1 16.3
Note. Following BF,/butanol transesterification, the alcohols were analyzed as the trimethylsilyl derivatives and the chain length was computed using the M+-CH3 ion fragment.
relative levels of fatty acid synthase and other protein bands showed relatively little change during this period. To determine whether the thioesterase gene transcript levels correlated with the thioesterase enzyme level, Northern blot analysis was performed with the thioesterase cDNA as the probe. A typical result is shown in Fig. 3. At all ages, when thioesterase gene transcripts were detected, the size of the transcript was 1.1 kb, as previously seen with adult mallards (23). The thioesterase transcript was detectable when the ducklings were 3 weeks old. The level of the transcript increased steadily with age until the maximum levels, similar to those found in adults, were observed when the birds were 2 months old (Fig. 3). DISCUSSION Uropygial gland lipid composition has been considered a good parameter for chemotaxonomic purposes (25,27). However, developmental and hormonal states of the animals could significantly influence the composition. Such a possibility was raised when it was observed that the diastereoisomer composition of the alkane-2,3-diols of the uropygial glands of chickens was developmentally regu-
TABLE Content Synthase
Duckling (days)
III
of Immunologically Measurable Thioesterase and Fatty Acid Glands of Developing Mallard
age
S-Acyl fatty acid synthase thioesterase” (bg/mg soluble
23 31 39 51 65 ’ Normal level for adult 105,OOOg supernatant.
Fatty acid synthase protein)
3.1 6.1 5.5 7.6 9.0 ducks
S-Acyl Fatty Acid Synthase in Uropygial Ducklings Soluble protein bs/s gland)
585 567 535 604 540 in noneclipse
is 12 pg/mg
50 42 32 23 26 protein
in
REGULATION 7
23
31
OF
THIOESTERASE
39 51 64
FIG. 3. Northern blot of duckling RNA probed with S-acyl fatty acid synthase thioesterase cDNA. RNA was isolated and 25 ag RNA of each sample were electrophoresed as described in the text. The probe used was full-length cDNA. Arrow indicates a molecular size of 1100 bp based on RNA molecular size markers. Numbers across the top indicate age of the ducklings in days.
lated (28). The results described in the present paper show that the composition of the uropygial gland lipids of mallards is developmentally regulated and that the shortchain wax esters, considered characteristic of mallards, become the dominant components of the gland only by the time the birds are 2 months old and have acquired the adult feather pattern. Until then, the glands contain long-chain esters similar to those found when adult mallards undergo postnuptial molt (eclipse). During mating season, dramatic changes occur in the composition of the uropygial gland lipids of female mallards. The monoester waxes are replaced completely with diesters of 3-hydroxy fatty acids (26) that have pheromonal properties (29). Thus it is clear that uropygial gland composition depends on the developmental stage and the hormonal state of the animal. Regulation of lipid synthesis in the uropygial glands is poorly understood. Lipid synthesis in the gland is regulated, most probably, by factors different from those involved in the more thoroughly studied organs such as the liver. For example, in both mallards (12) and geese (L. Rogers and P. E. Kolattukudy, 1982, unpublished observation), starvation and refeeding failed to show significant changes in the level of either acetyl-CoA carboxylase or fatty acid synthase in the uropygial gland. On the other hand, steroid hormone injections induced changes in the uropygial gland lipid composition (30) without inducing such changes in the liver. Since the sebaceous gland metabolism serves a unique function, it would not seem surprising that this process is regulated by a set of bioregulators different from those involved in other organs. The avian uropygial glands offer a convenient system to study such regulation. S-Acyl fatty acid synthase thioesterase, uniquely present in mammary glands (31, 32) and uropygial glands (9, 10) that produce short-chain acids, is responsible for the production of short-chain fatty acids in such tissues. Disappearance of short-chain wax esters from the mallard uropygial gland during postnuptial molt (eclipse) was found to be associated with a suppression of thioesterase gene expression, as manifested by drastically decreased
IN
UROPYGIAL
GLAND
IN
DUCK
205
levels of the enzyme and thioesterase transcript (4). During the developmental changes described in this paper, the appearance of the short-chain esters was correlated with the appearance of the thioesterase protein as measured immunologically and revealed by SDS-gel electrophoresis. The Northern blot analysis showed that the levels of thioesterase protein correlated with the appearance of thioesterase gene transcript. Thus it appears clear that the developmental changes in the chain length of the acyl portion of the wax are caused by the onset of expression of S-acyl fatty acid synthase thioesterase gene. Biochemical reasons for all of the observed changes in composition are not clear. The most obvious developmental change in the composition, other than the appearance of short-chain esters, is the drastic decrease in complexity of the composition. Until juvenile feathers began to appear 3 weeks after hatching, the waxes were composed of an extremely complex mixture of fatty acids with n-, mono-, and dimethyl branched fatty acids with little dominance of any component. With the acquisition of the adult feather pattern, the complexity decreased to the extent that monomethyl-branched acids became the dominant components, constituting 94% of the total acyl portion. Calculations showed that the proportion of the methyl branches in the acyl portion did not change with age. Thus the proportion of methylmalonyl-CoA and malonyl-CoA actually used in the production of the acyl chain appears to have remained reasonably constant during development. The release of shorter chain acids by the thioesterase would explain the dominance of the shortchain branched acids. However, the presence of longer 2and 4-methyl branched fatty acids cannot be easily explained. Since the nature of the enzymes that generate 2- and 4-methyl fatty acids is not clear, how only a single branch at either the 2- or 4- position is introduced remains obscure. Hormonal balance is probably involved in bringing about the change from long-chain esters to short-chain esters as the bird becomes an adult. However, little is known about how hormones regulate the uropygial gland metabolism. Injection of estradiol into mallards caused dramatic changes in the lipid composition of the mallard uropygial glands (30). This treatment caused a replacement of short-chain wax esters with long-chain esters. This observation suggested that hormonal balance can change the chain length. In fact, the first intron of the S-acyl fatty acid synthase thioesterase gene contains a cluster of putative steroid hormone receptor binding domains that could be involved in regulating the gene expression (33). Presumably, the androgen/estrogen balance that might be required for the production of shortchain wax esters is achieved when the birds reach 2 months of age. However, direct evidence to support this hypothesis is not available. During embryonic development of the uropygial gland, the genes characteristic of the gland (or uniquely expressed in the gland) begin expression a few days before
206
KOLATTUKUDY
hatching. For example, genes for cytoplasmic malonylCoA decarboxylase, responsible for causing the production of branched fatty acids characteristic of goose (ll), and malic enzyme and fatty acid synthase needed for the production of lipids in the duck uropygial glands (12) begin to be expressed 4 days before hatching. Obviously, transcriptional regulatory mechanisms involved in causing the gland-specific expression of the gene are set in motion several days before hatching so that the animal can produce the specialized lipids as soon as it emerges from the egg. On the other hand, the thioesterase gene responsible for the production of short-chain fatty acids is expressed only when the adult plumage is acquired by the mallard. Later in the life of the animal, the thioesterase gene is turned off during eclipse, again to be turned on at the end of eclipse. Thus, the regulation of expression of this thioesterase gene is probably regulated in the mallard gland in a way different from that of the cytoplasmic malonyl-CoA decarboxylase gene that appears to be turned on permanently several days before hatching. The regulatory mechanisms involved are not known and are currently under investigation in this laboratory.
ET
11. Kolattukudy, P. E., Rogers, L. M., Poulose, A. J., Jang, S. H., Kim, Y. S., Cheesbrough, T. M., and Liggitt, D. H. (1987) Arch. Biochem. Biophys. 256, 446-454. 12.
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P. E. (1988)
DNA
OF BIOCHEMISTRY
Vol. 284, No. 1, January,
AND
BIOPHYSICS
pp. 201-206,199l
Developmental Changes in the Expression of S-Acyl Fatty Acid Synthase Thioesterase Gene and Lipid Composition in the Uropygial Gland of Mallard Ducks (Anas platyrhynchos)’ P. E. Kolattukudy,’
Stewart
Ohio State Biotechnology
Center, The Ohio State University,
Received
July
18, 1990, and in revised
Bohnet,
form
Glenn
September
Sasaki,
and Linda Columbus,
14, 1990
Developmental changes in the composition of the uropygial gland secretory lipids of the postembryonic mallard ducks (Anasplatyrhynchos) were determined. During the first 3 weeks after hatching, the composition of the secretory lipids remained constant; the lipids consisted of long-chain wax esters composed of a complex mixture of n-, monomethyl, and dimethyl fatty acids esterified to n-Cl6 and n-Cl8 fatty alcohols. Afterward, as the ducks began to acquire adult feathers, short-chain wax esters composed of 2- and 4-monomethyl fatty acids began to appear with 2-methylhexanoyl and 4-methylhexanoyl as the major acyl components; esters of shortchain monomethyl fatty acids (
‘This work was supported, in part, by Grant GM-18278 from the National Institutes of Health. ’ To whom correspondence should be addressed at Ohio State Biotechnology Center, 206 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210. 0003.9861/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Rogers
Ohio 43210
Sebaceous glands produce unique lipids that serve a variety of functions on the animal surface (1,2). In birds a single large gland, called the uropygial gland, performs much the same functions as those of the numerous diminutive sebaceousglands that are widely distributed on the surface of mammals. The avian glands that are structurally and functionally analogous to the mammalian sebaceous glands present a convenient model for biochemical and molecular biological studies on this holocrine system (3-6). The synthesis of the unique lipids in the uropygial gland involves some unique proteins that function in conjunction with the usual enzymes. For example, in the goose uropygial gland, a unique cytoplasmic malonyl-CoA decarboxylase causes the production of multiple methyl branched fatty acid by removing malonyl-CoA to ensure that methylmalonyl-CoA is the only substrate available to the fatty acid synthase that synthesizes the branched acids when only the branched precursor is available (68). In the mallard duck uropygial gland, a unique 30-kDa thioesterase hydrolytically releasesshort-chain fatty acids from fatty acid synthase and thus causes production of short-chain wax esters (9, 10). Initiation of the synthesis of these tissue-specific proteins is a biochemical manifestation of the differentiation of the gland. Expression of the genes that code for such unique proteins would provide a useful parameter for studying the developmental biology of such sebaceous glands. In developing embryonic goose uropygial glands, malonyl-CoA decarboxylase transcripts appeared several days prior to hatching and reached maximal levels by hatching (11). In the developing duck embryonic uropygial glands, malic enzyme and fatty acid synthase transcripts increased dramatically several days before hatching (12). High levels of these enzymes are required for the production of the gland lipids, although these enzymes are not unique to the glands. The developmental pattern of 201
202
KOLATTUKUDY
ET
AL.
expression of the tissue-specific thioesterase gene involved in the production of the unique short-chain lipids of the gland has not been studied. In this paper we report that the expression of this gene occurs only when the mallards acquire adult feathers, replacing their down feathers. The composition of the uropygial gland lipids also reflects this change in gene expression by the replacement of longchain wax esters by the short-chain wax esters that usually are considered characteristic of mallards. MATERIALS
AND
METHODS
Materials. Ducklings were purchased from Lind’s Hatchery in Umatilla, Oregon, or from Whistling Wings in Hanover, Illinois. They were maintained in outdoor pens on standard chick mash (minus antibiotics). Dithioerythritol (DTE)3 and N,O-(trimethylsilyl)acetamide were purchased from Sigma. [iz51] Protein A (80-90 Ci/g) was from New England Nuclear. BFB (14%) in butanol was prepared by slowly bubbling BFB gas into freshly distilled n-butanol until 14% weight gain was obtained. All restriction enzymes were purchased from New England Biolabs. Nitrocellulose and Nytran membranes were from Schleicher & Schuell. Young birds (2, 7, Tissue preparations and extraction of products. and 14 days) were sacrificed by exsanguination and the uropygial glands were carefully dissected from the surrounding fat and connective tissue (3). Glands were divided into two portions: one was quickly frozen in liquid nitrogen and stored at -80°C for RNA and DNA analysis and the other was homogenized in chloroform:methanol (2:l). The lipids were recovered in chloroform according to the method of Bligh and Dyer (13) and the solvent was evaporated with a gentle stream of nitrogen. With older ducklings (3 weeks or more) the waxes were collected by gently squeezing the gland to express the lipids into a test tube where they were stored at -2O’C until further analysis. At Days 23, 31, 39, 51, and 65, two birds were sacrificed and portions of each gland were used for protein characterization and RNA and DNA extraction. Intact wax was injected into a HewlettAnalysis of the into& wan. Packard Model 5840A gas chromatograph equipped with a 10 m X 0.2 mm OV-1 capillary column and attached to a HP5985 mass spectrometer to obtain electron impact mass spectra of the intact waxes. Column temperature was set at 15O’C for 1 min, followed by a 150-280°C temperature program at lO”C/min. Mass spectra were recorded with 70 eV ionizing voltage. Wax esters were identified by their diagnostic ions representing the fatty acid portion (RCOOH:) (4). The ions representing these fragments were quantitated and tabulated as relative percentages of the total under the selected ion display (SID) option of the mass spectrometer. This option allows for the simultaneous display and quantitation of selected ion profiles derived from the total ion data. All values were standardized by comparison to the values obtained with equimolar amounts of octadecyl heptanoate and octadecyl octadecanoate. Diester waxes were identified by using ions at m/e 143, 171, and 199, corresponding to the fragments of 3-hydroxy octanoate, -decanoate, and -dodecanoate, respectively. The values obtained as relative percentages for these fragments using the SID program were identical to the values obtained following analysis of the products of transesterification of the diesters with BFB in methanol. These ions were therefore used to identify and quantitate the intact diester waxes. Compositional analysis of the wax esters. Waxes were transesterified by refluxing with 14% BF, in butanol (14). The reaction products were plated on l-mm-thick silica gel G TLC plates and developed in hexane: ethyl ether:formic acid (9O:lO:l v/v). The butyl esters were eluted from the silica gel with chloroform:methanol (2:l) and analyzed by gas chromatography on an OV-1 column (10 m X 0.2 mm). The column was maintained at 150°C for 1 min. followed by a temperature program to
3 Abbreviations
used: DTE,
dithioerythritol;
SID, selected
ion display.
I
5
15 TIME C&d)
FIG. 1.
Gas-liquid chromatograms pygial glands of mallard ducklings. 65.day-old ducklings, respectively. scribed in the text.
of intact wax esters from the uroA, B, and C represent 23-, 39-, and Capillary GLC was performed as de-
280°C at lO”/min. The alcohol was recovered from the silica gel by elution with diethyl ether and incubated with bis-N,O-(trimethylsilyl)acetamide at 90°C for 30 min to convert the alcohol to the trimethysilyl ether. These derivatives were analyzed by GLC-MS using the same column and temperature conditions as those used for the analysis of the butyl esters. Uropygial glands were dissected from Enzyme level measurements. ducklings sacrificed at 23,31,39,51, and 65 days of age, quickly frozen in liquid nitrogen, and stored at -80°C for further analysis. For protein analysis, each sample was homogenized in 0.1 M sodium phosphate buffer, pH 7, containing 0.25 M sucrose and 1 mM DTE (3 ml buffer/g tissue). Homogenates were sequentially centrifuged 5 min at lOOOg, 15 min at 27,000g and 90 min at 105,OOOg; all pellets were discarded. Total protein in the 105,OOOg supernatant was measured by the method of Lowry et al. (15). Fatty acid synthase and S-acyl fatty acid synthase thioesterase in the soluble protein were quantitated by a [lz51] protein A-immunoblot method (16). Rabbit antibody against the synthase was prepared as previously described (17) and the antibodies against the thioesterase were prepared similarly using the purified duck thioesterase (10). SDSpolyacrylamide gel electrophoresis was performed following the method of Laemmli (18), using 10% resolving and 3% stacking gels. Isolation of RNA and genomic DNA. Portions (2 g) of uropygial glands and liver were ground into powder in liquid NP, and DNA and RNA were isolated from uropygial glands by the method of Zehner et al. (19) with the addition of ultracentrifugation through CsCl gradients. After the phenol extraction and ethanol precipitation the nucleic acids
REGULATION
OF
THIOESTERASE
were dissolved in 8 ml of 1 M CsCl containing 1% P-mercaptoethanol. The solution was layered over 5 ml of 5.7 M CsCl containing 1% 2mercaptoethanol and spun at 22,000 rpm overnight in a Beckman SW28 rotor. The DNA layer (detected by its Schlieren pattern) was removed by syringe and the RNA pellet was dissolved in 10 mM Tris-HCl, pH 7.6, containing 1% 2-mercaptoethanol and 1 mM EDTA and was then ethanol precipitated. The DNA layer was mixed with CsCl to a final solution density of 1.42; 100 ~1 of 10 mg/ml ethidium bromide was added and the solution was centrifuged at 55,000 rpm overnight in a Beckman Ti70 rotor. The DNA layer was removed by syringe, butanol extracted, and dialyzed against 10 mM Tris-HCl, pH 7.6, containing 1 mM EDTA. RNA was dissolved in diethylpyrocarbonate-treated H,O. Northern blot analysis. Electrophoresis of 25 Kg RNA from each sample was run on a 1.2% agarose-formaldehyde gel overnight at 25 V (20) with RNA standards from BRL Life Technologies, Inc. The RNA was blotted onto Nytran via capillary action using 10 mM NaOH as the transfer medium (21). The blot was baked for 2 h at 80°C and hybridized with 3zP nick translated (22) S-acyl fatty acid synthase thioesterase cDNA (23) in formamide/Denhardt’s-based solution at 42’C (24).
RESULTS
Short-chain wax esters constitute the bulk of secretory lipids produced by adult mallards (25). However, gas-liquid chromatograms of intact lipids collected from ducklings of ages 2 to 21 days showed a pattern quite different from that of the normal adult. Long-chain wax esters were the major components in the ducklings (Fig. 1A). After 3 weeks of age, the GLC pattern from the intact waxes gradually changed to include components with shorter retention times (Fig. 1B). As the adult feather patterns emerged, the uropygial gland products attained a pattern identical to that of the mature mallard ducks (Fig. 1C). Diesters of 3-hydroxy fatty acids were minor components and at no time did they constitute greater than 10% of the total lipids. Analysis of the components of the diesters showed that the composition was quite similar to that previously reported (26). To determine fatty acid composition, the intact waxes from the uropygial glands of ducklings were transesterified with BFB in butanol and the butyl esters were analyzed by gas-liquid chromatography and mass spectrometry. The waxes of the hatched ducklings contained a very complex mixture of fatty acids (Table I). The pattern remained constant until the ducklings were 23 days old. The fatty acids from the glands of ducklings of 1 to 23 days of age contained a high proportion of multimethylbranched fatty acids and were longer than those found in adults. As the ducklings became older the fatty acids produced by the gland became progressively shorter and the content of multimethyl-branched acids decreased. The fatty acid composition of the wax from the 31-day-old duckling showed a transition to the more adult-like composition reached by 65 days of age, as shown in Table I. At 2 months, when adult feathers became the dominant covering of the birds, the characteristic monomethylbranched Cs acids produced by the adult mallards became a dominant component of wax esters. The most striking change in the fatty acid composition was that short-chain acids, characteristic of the adult mallard, appeared as the dominant components only at
TN
UROPYGIAL
GLAND
IN
203
DUCK
TABLE
I
Developmental Changesin the Composition of the Acyl Portion of the Uropygial Gland Wax of Ducklings % Composition Fatty
acid
Age in days:
2-Me-C, 4-Me-C, 2-Me-C!? 4-Me-C, 2-Me-C, 4-Me-Cs 2,6-Me.& 2-Me-& 4-Me-C, 2-Me-Cl0 4-Me-C0 2,6-MeZ-C,0 n-Cl1 2-Me-C1 2,6-Me&,, 4-Me-C,, 2-Me-t& 4-Me-f& 2,6-Me&, n-C14 4,8-Me&& 2-Me-f& 4-Me-Cl3 2,6,8-Me&i3 2,8-Me&s 2,6,10-Me&i3 n-C15 4,6-Me&i3 2-Me-C,, 4-Me-‘& 2,6-Me&, 4,6-Me&i4 4,8-Me&, 4,10-MeZ-C11 4-Me-& 2,6,8-Me&, 4,12-Me&,, 2-Me-& 4-Me-C& 4,6-Me&, 2,10-Me2-Cn 2-Me-Cl6 n-C16 2-Me-C,, 4-Me-Cl7 2,6-Me&s 4,6-Me&& 2,6-Me&i7 48Me&s 4,10-Me&i6 4,12-Me&,, 28Me&r 4,8-Me&i7 4-Me-C,s 4-Me-C& Total
23
31
65
0.13 0.22
16.45 24.86 3.29
0.20 0.06 0.30
0.36 2.83
3.50 8.31 10.10
1.70 3.88 0.32 0.23
6.03 6.33 0.58 0.61
1.14 0.20 0.33 1.41 1.87 1.66
1.01 3.09
8.15 9.27
6.37 4.53
1.24 1.38
1.36 0.37 1.98
1.65 0.83 1.27 1.24 5.65 2.65 1.30 2.11 2.70 2.68 1.76
2.27 2.38 1.37
0.91
1.63 5.48 7.54 1.83 3.02
3.28 3.53
1.92
1.87 1.27 4.48 1.37 2.43 0.89
2.217 2.49
2.18
2.84
0.80 5.12
2.18 2.00
5.79
2.06 2.02 2.53 3.79
3.14 3.20 1.89
2.24 1.41 0.96 1.04
0.97
0.68 2-Methyl 4-Methyl
18.37 11.89
25.75 34.78
41.58 52.58
204
KOLATTUKUDY
ET
AL. TABLE
II
The Alcohol Portion of the Wax Esters from Uropygial Glands of Developing Mallard Ducklings 5% Composition Chain length
E t i!
0
59
Day from Hatch FIG. 2. Changes in the composition of the acyl portion of the wax esters from the uropygial glands of developing mallard ducklings. Shortchain fatty acids are defined as Cl2 or less. Fatty-acid chain length was determined by GLC-MS analysis as described in the text.
2 months of age. Until after 3 weeks of age, only about 10% of the fatty acids were of short-chain length (
Age in days:
Cl6 Cl7 CL3 Cl9 C 20
23
31
39
65
0.8 22.1 55.4 11.0 10.0
1.9 27.6 47.2 13.6 9.8
2.0 43.5 38.5 10.9 5.2
4.2 36.3 31.1 12.1 16.3
Note. Following BF,/butanol transesterification, the alcohols were analyzed as the trimethylsilyl derivatives and the chain length was computed using the M+-CH3 ion fragment.
relative levels of fatty acid synthase and other protein bands showed relatively little change during this period. To determine whether the thioesterase gene transcript levels correlated with the thioesterase enzyme level, Northern blot analysis was performed with the thioesterase cDNA as the probe. A typical result is shown in Fig. 3. At all ages, when thioesterase gene transcripts were detected, the size of the transcript was 1.1 kb, as previously seen with adult mallards (23). The thioesterase transcript was detectable when the ducklings were 3 weeks old. The level of the transcript increased steadily with age until the maximum levels, similar to those found in adults, were observed when the birds were 2 months old (Fig. 3). DISCUSSION Uropygial gland lipid composition has been considered a good parameter for chemotaxonomic purposes (25,27). However, developmental and hormonal states of the animals could significantly influence the composition. Such a possibility was raised when it was observed that the diastereoisomer composition of the alkane-2,3-diols of the uropygial glands of chickens was developmentally regu-
TABLE Content Synthase
Duckling (days)
III
of Immunologically Measurable Thioesterase and Fatty Acid Glands of Developing Mallard
age
S-Acyl fatty acid synthase thioesterase” (bg/mg soluble
23 31 39 51 65 ’ Normal level for adult 105,OOOg supernatant.
Fatty acid synthase protein)
3.1 6.1 5.5 7.6 9.0 ducks
S-Acyl Fatty Acid Synthase in Uropygial Ducklings Soluble protein bs/s gland)
585 567 535 604 540 in noneclipse
is 12 pg/mg
50 42 32 23 26 protein
in
REGULATION 7
23
31
OF
THIOESTERASE
39 51 64
FIG. 3. Northern blot of duckling RNA probed with S-acyl fatty acid synthase thioesterase cDNA. RNA was isolated and 25 ag RNA of each sample were electrophoresed as described in the text. The probe used was full-length cDNA. Arrow indicates a molecular size of 1100 bp based on RNA molecular size markers. Numbers across the top indicate age of the ducklings in days.
lated (28). The results described in the present paper show that the composition of the uropygial gland lipids of mallards is developmentally regulated and that the shortchain wax esters, considered characteristic of mallards, become the dominant components of the gland only by the time the birds are 2 months old and have acquired the adult feather pattern. Until then, the glands contain long-chain esters similar to those found when adult mallards undergo postnuptial molt (eclipse). During mating season, dramatic changes occur in the composition of the uropygial gland lipids of female mallards. The monoester waxes are replaced completely with diesters of 3-hydroxy fatty acids (26) that have pheromonal properties (29). Thus it is clear that uropygial gland composition depends on the developmental stage and the hormonal state of the animal. Regulation of lipid synthesis in the uropygial glands is poorly understood. Lipid synthesis in the gland is regulated, most probably, by factors different from those involved in the more thoroughly studied organs such as the liver. For example, in both mallards (12) and geese (L. Rogers and P. E. Kolattukudy, 1982, unpublished observation), starvation and refeeding failed to show significant changes in the level of either acetyl-CoA carboxylase or fatty acid synthase in the uropygial gland. On the other hand, steroid hormone injections induced changes in the uropygial gland lipid composition (30) without inducing such changes in the liver. Since the sebaceous gland metabolism serves a unique function, it would not seem surprising that this process is regulated by a set of bioregulators different from those involved in other organs. The avian uropygial glands offer a convenient system to study such regulation. S-Acyl fatty acid synthase thioesterase, uniquely present in mammary glands (31, 32) and uropygial glands (9, 10) that produce short-chain acids, is responsible for the production of short-chain fatty acids in such tissues. Disappearance of short-chain wax esters from the mallard uropygial gland during postnuptial molt (eclipse) was found to be associated with a suppression of thioesterase gene expression, as manifested by drastically decreased
IN
UROPYGIAL
GLAND
IN
DUCK
205
levels of the enzyme and thioesterase transcript (4). During the developmental changes described in this paper, the appearance of the short-chain esters was correlated with the appearance of the thioesterase protein as measured immunologically and revealed by SDS-gel electrophoresis. The Northern blot analysis showed that the levels of thioesterase protein correlated with the appearance of thioesterase gene transcript. Thus it appears clear that the developmental changes in the chain length of the acyl portion of the wax are caused by the onset of expression of S-acyl fatty acid synthase thioesterase gene. Biochemical reasons for all of the observed changes in composition are not clear. The most obvious developmental change in the composition, other than the appearance of short-chain esters, is the drastic decrease in complexity of the composition. Until juvenile feathers began to appear 3 weeks after hatching, the waxes were composed of an extremely complex mixture of fatty acids with n-, mono-, and dimethyl branched fatty acids with little dominance of any component. With the acquisition of the adult feather pattern, the complexity decreased to the extent that monomethyl-branched acids became the dominant components, constituting 94% of the total acyl portion. Calculations showed that the proportion of the methyl branches in the acyl portion did not change with age. Thus the proportion of methylmalonyl-CoA and malonyl-CoA actually used in the production of the acyl chain appears to have remained reasonably constant during development. The release of shorter chain acids by the thioesterase would explain the dominance of the shortchain branched acids. However, the presence of longer 2and 4-methyl branched fatty acids cannot be easily explained. Since the nature of the enzymes that generate 2- and 4-methyl fatty acids is not clear, how only a single branch at either the 2- or 4- position is introduced remains obscure. Hormonal balance is probably involved in bringing about the change from long-chain esters to short-chain esters as the bird becomes an adult. However, little is known about how hormones regulate the uropygial gland metabolism. Injection of estradiol into mallards caused dramatic changes in the lipid composition of the mallard uropygial glands (30). This treatment caused a replacement of short-chain wax esters with long-chain esters. This observation suggested that hormonal balance can change the chain length. In fact, the first intron of the S-acyl fatty acid synthase thioesterase gene contains a cluster of putative steroid hormone receptor binding domains that could be involved in regulating the gene expression (33). Presumably, the androgen/estrogen balance that might be required for the production of shortchain wax esters is achieved when the birds reach 2 months of age. However, direct evidence to support this hypothesis is not available. During embryonic development of the uropygial gland, the genes characteristic of the gland (or uniquely expressed in the gland) begin expression a few days before
206
KOLATTUKUDY
hatching. For example, genes for cytoplasmic malonylCoA decarboxylase, responsible for causing the production of branched fatty acids characteristic of goose (ll), and malic enzyme and fatty acid synthase needed for the production of lipids in the duck uropygial glands (12) begin to be expressed 4 days before hatching. Obviously, transcriptional regulatory mechanisms involved in causing the gland-specific expression of the gene are set in motion several days before hatching so that the animal can produce the specialized lipids as soon as it emerges from the egg. On the other hand, the thioesterase gene responsible for the production of short-chain fatty acids is expressed only when the adult plumage is acquired by the mallard. Later in the life of the animal, the thioesterase gene is turned off during eclipse, again to be turned on at the end of eclipse. Thus, the regulation of expression of this thioesterase gene is probably regulated in the mallard gland in a way different from that of the cytoplasmic malonyl-CoA decarboxylase gene that appears to be turned on permanently several days before hatching. The regulatory mechanisms involved are not known and are currently under investigation in this laboratory.
ET
11. Kolattukudy, P. E., Rogers, L. M., Poulose, A. J., Jang, S. H., Kim, Y. S., Cheesbrough, T. M., and Liggitt, D. H. (1987) Arch. Biochem. Biophys. 256, 446-454. 12.
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Bligh, E. G., and Dyer, 911-917.
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