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Isolation of a goat acetyl-coa carboxylase complementary DNA and effect of milking frequency on the expression of the acetyl-coa carboxylase and fatty acid synthase genes in goat mammary gland
Comp. Biochem. PhysioL Vol. 105B,No. 1, pp. 123-128, 1993 Printed in Great Britain
0305-0491/93$6.00+0.00 © 1993PergamonPress Ltd
ISOLATION OF A GOAT ACETYL-COA CARBOXYLASE COMPLEMENTARY D N A A N D EFFECT OF MILKING FREQUENCY ON THE EXPRESSION OF THE ACETYL-COA CARBOXYLASE AND FATTY ACID SYNTHASE GENES IN GOAT MAMMARY GLAND MAUREEN T. TRAVERS and MICHAEL C. BARBER*
Hannah Research Institute, Ayr, KA6 5HL, Scotland, U.K. (Tel. 0292-76013; Fax 0292-671052) (Received 28 August 1992; accepted 11 November 1992)
Abstract--1. Using the polymerase chain reaction we have isolated a partial complementary DNA for goat acetyl-CoA carboxylase which is 90 and 82% homologous to the published rat and chicken complementary DNA sequences, respectively. 2. Frequent milking causes an upregulation of the acetyl-CoA carboxylase and fatty acid synthase genes in goat mammary gland that parallels the increase in the respective enzyme activities. 3. The sequence for goat acetyl-CoA carboxylase is in the EMBL data base, Accession Number Z17803.
INTRODUCTION
It is well established that milking dairy animals more frequently than the usual twice a day increases milk yield. This effect in the goat is both acute (within hours; Henderson et al., 1983) and chronic (after weeks; Henderson et al., 1985) and can be accounted for by immediate increases in the secretory activity of the mammary cells and by more long-term adaptations resulting initially in increased cellular activity followed by increases in the mammary cell population (Wilde et al., 1987). Several studies (Wilde et al., 1987; Knight et al., 1990) have shown that a consequence of long-term frequent milking is a cellular up-regulation of a number of enzymes of the lipogenie pathway, notably acetyl-CoA carboxylase (ACC EC 6.4.1.2) and fatty acid synthase (FAS EC 2.3.1.85). The mechanism of this up-regulation is unknown though it is thought to relate, in part, to an increased rate of removal of an inhibitory autocrine factor in the more frequently milked gland (Wilde and Peaker, 1990). Recently, a 10-30kDa whey fraction purified from goat milk has been shown to inhibit the induction of FAS protein in cultures of mid-pregnant mouse mammary cells cultured on floating collagen gels (Wilde et al., 1991). In this present study we describe the cloning of a complementary DNA (eDNA) for goat ACC and show, using this eDNA together with a human FAS eDNA (Chalbos et al., 1987), that increased milking frequency results in increases in ACC and FAS gene
*To whom correspondence should be addressed.
Abbreviations--PCR: Polymerase chain reaction; bGH: recombinant bovine growth hormone; RNase: ribonuelease.
expressions that parallel the cellular increases in the enzyme activities. This suggests that the relief from chemical feedback inhibition in the more frequently milked gland results in increased ACC and FAS enzyme activity at least partly through a pre-translational mechanism, possibly through increases in the transcription rate or stabilization of the messenger RNAs (mRNA), or both. MATERIALS AND METHODS
Animals
British Saanen goats from the Hannah Research Institute herd were used in their first-to-third lactation, as described previously (Knight et al., 1990). Ten goats at approximately the same stage of lactation and of similar body weight (70.6 + 1.1 kg, S E M ) were paired for milk yield (overall mean of 23.2 + 1.48 kg/week). At week 19 of lactation, one goat of each pair was injected daily with 0.15 mg recombinant bovine growth hormone (bGH)/kg and the other goat received the vehicle only, as described previously (Knight et al., 1990). Starting at the same time as the daily injections the right mammary gland of each goat was switched from twice- to thrice-daily milking (07.30, 15.30 and 23.30hr); the left gland remained on twice-daily milking (0.730 and 15.30hr). The experiment continued for 22 weeks at the end of which time the goats were killed and samples of mammary tissue were quickly excised and frozen in liquid nitrogen. Cloning o f a goat acetyl-CoA carboxylase eDNA by the polymerase chain reaction
Poly (A ÷ ) RNA was prepared from total cellular RNA (Chirgwin et al., 1979) using oligo (dT) 123
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MAUREENT. TRAVERSand M1cr~.~LC. BARBER
cellulose chromatography (Aviv and Leder, 1972). For the preparation of first strand eDNA, 1/~g of poly (A + ) RNA was denatured and then annealed to 5 #g random hexamers (Pharmacia, Uppsala, Sweden). The final reaction conditions were 50/~g/ml poly (A +) RNA, 250#g/ml random hexamers, 50raM Tris-HC1, pH8.3, 50mM KC1, 10mM MgC! 2, 1 mM EDTA, 10/~g/ml BSA, 1 mM DTT, 0.5 mM spermidine, 1.5 mM of each deoxynucleoside triphosphate, 1000U/ml RNAguard (Pharmacia), 150/~Ci/ml~ [32p]dCTP (3000 Ci/mmol, Amersham, Bucks, U.K.) and 250U/ml avian myeloblastosis virus reverse transcriptase (Pharmacia) in a total volume of 20 #1. The reaction was incubated at 42°C for 90 min. EDTA and NaOH were added to give a final concentration of 20 mM and 0.2 M respectively and the mixture incubated at 60°C for 1 hr. The eDNA was precipitated by addition of 0.1 vol 3M sodium acetate, pH 6.0, and 2 vol absolute ethanol. The efficiency of the reaction was determined by the incorporation of the [32p] dCTP into the eDNA and was generally in the range 25-30% of input RNA. To amplify a cDNA sequence corresponding to goat acetyl-CoA carboxylase 50 ng first strand eDNA was added to 100#1 of Polymerase Chain Reaction (PCR) buffer (10% v/v dimethyl sulphoxide, 67mM TrisHCI, pH 8.8, 7.6mM MgC12, 16.6mM (NH4)2SO4, 1 mM 2-mercaptoethanol, 67/~M EDTA, 170/~g/ml bovine serum albumin and 1.5mM each deoxynucleoside trophosphate (Frohman, 1990) containing 100 pmol each of the oligodeoxynucleotide upstream and downstream primers. The oligodeoxynucleotides were synthesized on an Applied Biosystems 81A DNA synthesizer. The upstream primer (5'GAAAACCCAGATGAGGGGTTTAAGCCCAG 3') and downstream primer (5' AGTCATTACCATCTTCATTACCTCAATCTCTGCATA 3') were complementary to the 3' ends of the double-stranded eDNA sequence encompassing amino acid residues 512-789 of the coding region of rat mammary gland acetyl-CoA carboxylase (Lopez-Casillas et al., 1988). The mixture was denatured at 95°C for 5 min and after cooling to 72°C, 2.5 units Thermus aquaticus (Taq) DNA polymerase (Anglian Biotec. Ltd, Colchester, UK) were added. The reaction mixture was then subjected to 45 cycles of denaturing at 92°C for 30 sec, annealing of the primers at 40°C for 30 sec and extending the eDNA at 72°C for 1 min. The resulting PCR product was phenol/chloroform extracted, the 3' ends flushed with T4 DNA polymerase (Davis et al., 1986) and cloned into the pGEM 3zf (___) (Promega) series of phagemid vectors. Singlestranded DNA was produced from the pGEM 3zf ( + ) phagemids by infection of recombinant JM 109 E. coli with R408 Helper phage (Russel et al., 1986). Both strands of the cloned PCR product was sequenced using the dideoxy chain termination method (Sanger et al., 1977) with the aid of sequencing grade Taq DNA polymerase (Promega) (Innis et al., 1988).
Northern blot analysis o f R N A and enzyme measurements
Forty micrograms of total cellular RNA was separated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde and then transferred to hybond N nylon membranes (Amersham) (Davis et al., 1986). For the measurement of FAS mRNA abundance we obtained a human FAS eDNA (H. Rochefort, INSERM, France; Chalbos et al., 1987). The human FAS eDNA insert was sub-cloned into pGEM 3zf ( + ) and a labelled antisense RNA probe was generated from the SP6 promoter (Melton et al., 1984). For the measurement of ACC mRNA the cloned ACC PCR product was labelled to high specific activity by random priming (Feinberg and Vogelstein, 1983). Membranes were hybridized at 42°C (ACC eDNA probe) or 55°C (FAS riboprobe) overnight in hybridization solution (50% formamide, 0.75 M NaCI, 50 mM sodium dihydrogen phosphate, pH 7.4, 5 mM EDTA and Ficoll 400, bovine serum albumin, polyvinylpyrolidone and SDS each at 0.1% (w/v), and 200/~g of denatured salmon testes DNA/ml containing labelled probe at 2 x 10 6 dpm/ ml. After hybridization the membranes were washed in 15mM NaCI, 1.5mM sodium citrate, 0.1% w/v SDS at 50-55°C. An RNase digestion step was included before the final wash of membranes hybridized with the antisense FAS RNA probe. Membranes were exposed to XAR-5 film at - 7 0 ° C with intensifying screens. The autoradiographs were scanned using a Biorad laser densitometer with 1-D software. Total ACC and FAS enzyme activities were collated from previously published data (Knight et al., 1990). Statistical analysis
Differences were assessed by analysis of variance fitting the effects of twice- vs thrice-daily milking alone, the effect of bGH alone and the interaction between bGH and differential milking. As the effects o f b G H or the interaction between bGH and differential milking never approached significance (P in the range 0.75-0.90) data from bGH-treated goats and control goats was pooled and the results have been expressed in terms of the effects of twice- vs thricedaily milking. RESULTS
Amongst the variety of purposes that PCR has been applied to is the cross-species isolation of homologous genes (Erlich et al., 1991). A common feature of most applications of PCR is the use of primers designed to match two known or presumed eDNA or genomic DNA sequences. Previous to this present study the eDNA sequences for chicken (Takai et al., 1988) and rat (Lopez-Casillas et al., 1988) acetylCoA carboxylase have been reported. Comparison of the coding region of the two acetyl-CoA carboxylase cDNAs shows them to be 80 and 96% homologous
125
Mammary gland lipogenic enzyme gene expression in eDNA sequence and amino acid sequence, respectively, in a region 1 kb upstream of the biotin binding site. In this present study, by applying a strategy of using relatively long primers derived from the rat ACC eDNA that show greater-than-average sequence homology with the homologous chicken ACC eDNA, we have been able to amplify an 834bp cDNA from lactating goat mammary gland eDNA. Because the downstream primer corresponds to the region coding for the biotin-binding site of the protein the 834 bp cDNA encompasses 768 bp of novel sequence just upstream of the putative biotin-binding site and is of the predicted size by comparison with
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the homologous rat and chicken cDNAs. DNA sequence analysis of this goat ACC eDNA, which corresponds to about 11% of the eDNA encompassing the putative coding region, shows it to be 90 and 82°/. homologous to the corresponding rat and chicken eDNAs, respectively; the putative amino acid sequence is 96.1 and 94.5% homologous, respectively (Fig. 1). Amplification of rat mammary gland eDNA with the two PCR primers yielded a sequence 99.6% homologous to the published rat ACC sequence (Lopez-Casillas et aL, 1988) and use of this rat ACC eDNA in Northern analysis of rat mammary gland and adipose tissue RNA has shown a marked
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Fig. 1. Partial nucleotide sequence of goat acetyl-CoA carboxylase obtained from PCR amplification. The deduced amino acid sequence for goat acetyl-CoA carboxylase is displayed below the nucleotide sequence in one letter code together with the previously published amino acid sequences for rat (Lopez-Casillas et aL, 1988) and chicken (Takai et aL, 1988) acetyl-CoA carboxylase for comparison. The numbers correspond to the amino acid notation in rat acetyl-CoA carboxylase (Lopez-Casillas et aL, 1988).
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MAUREEN T. TRAVERS and MICHAEL C. BARBER
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Fig. 2. Northern blot analysis of 40 #g total cellular RNA from a lactating goat milked three times (3 × ) a day on one mammary gland and twice a day (2 x ) on the other for 22 weeks. (A) Hybridization with goat ACC cDNA probe. (B) Hybridization with the human FAS riboprobe. The 11.5 kb ACC mRNA and the 10.7 kb FAS mRNA were integrated by densitometry (data summarized in Table 1).
concordance with ACC enzyme activities, suggesting transcriptional control in these rat tissues (Barber et al., 1992). Northern blot analysis (Fig. 2A) showed hybridization of the ACC cDNA probe to a major mRNA species of 11.5 kb in lactating goat mammary tissue, which is similar in size to the reported rat ACC mRNA (Bai et aL, 1986). A minor hybridizing species of around 3.8 kb is also observed with the goat ACC cDNA probe. This species has also been observed in RNA from rat tissues and has been attributed to degraded ACC mRNA (Bai et al., 1986). In lactating rats subjected to a variety of endocrinological manipulations, the abundance of the major ACC mRNA and of the 3.8 kb species in mammary and adipose tissue RNA varied in parallel with changes in total A C C enzyme activity, giving some support to this assertion (Barber et al., 1992). The 11.5 kb species represents the most functionally active species and therefore, in this present study, this has been primarily considered in the comparisons that have been made between total enzyme activity and functional acety142oA carboxylase gene expression. Conversely, the human FAS riboprobe hybridized to an mRNA species of approximately 10kb (Fig. 2B) which is slightly larger than the reported size for the human mRNA of 8 kb (Chalbos et al., 1987). Use of the ACC cDNA and FAS riboprobe in experiments to assess the effects of differential milking on ACC and FAS gene expression in lactating goat mammary gland showed that prolonged thricedaily milking produced a marked increase (P <0.01) in the amount of ACC and FAS mRNA/mg DNA compared with twice daily milking and this paralleled the increases observed for ACC and FAS enzyme activity/mg DNA (Table 1). This effect appears to be
achieved in two ways in that thrice-daily milking not only increases (P < 0.05) the abundances of ACC and FAS mRNAs per unit total cellular RNA (Fig. 2; Table 1) but also results in a small but significant (P <0.05) increase in the amount of RNA per cell (Table 1; Wilde et al., 1987). This suggests that the effect of thrice-daily milking to overcome chemical feedback inhibition is both specific in terms of its effect on ACC and FAS gene expression but also more general in terms of the increase of the RNA content per mammary cell. DISCUSSION
The present study extends the observations of Knight et al., (1990) to show that the increase in total Table 1. Effect of milkingfrequencyon ACC and FAS gene expression and enzymeactivitiesin goat mammarygland Milkingfrequency 2x 3× mg RNA/mg D N A
ACC m R N A abundance U/40 # g R N A FAS m R N A abundance U/40 # g R N A Units ACC mRNA/mg DNA Units FAS mRNA/mg DNA Enzyme activity~" ACC (nmol/mio/mg DNA) FAS (nmol/min/mg DNA)
0.77 _+ 0.09
1.00 _+ 0.08*
0.73 _+_0.24
1.74 + 0.52*
2.57 _+ 0.48
4.54 _+ 0.88*
16.6 _+ 6.5
41.0 _+ 3.2**
53.4 _+ 14.3
114.0 _+ 23.0**
73.7 _+ 8.9 453 + 85
97.0 _+ 6.7** 774 _+ 75**
Total cellular RNA was isolated from goat mammaryglands differentiallymilkedfor 22 w~eks.AcetyI-CoAcarboxylase and fatty acid synthasemRNA abundancewas determined by Northern analysisand has been express~ as the integrated area of the 11.5kb ACCmRNAand the 10.7kb FAS mRNAnormalizedagainst28s ribosomalRNA. Each point is the mean +_ SEM of 10 animals.
*P<0.05, **P<0.01 compared with 2 x gland. ~'Data from Knight et al., (1990).
Mammary gland lipogenic enzyme gene expression ACC and FAS enzyme activities associated with thrice-daily milking is caused in part by an increase in the abundances of the mRNAs for ACC and FAS. However, these observations do not preclude the importance of other mechanisms such as the control of mRNA translation and enzyme degradation (Majerus and Kilburn, 1969) and control of enzyme activity by reversible phosphorylation (Kim et al., 1989) which undoubtedly play a role in the lipogenic potential of the tissue. Though the increase in the abundances of the ACC and FAS mRNAs may be due to an increase in the transcription rate or an increase in mRNA stabilization, or a combination of both, the means by which the effect overall is achieved is a matter of conjecture. Previous studies have shown that the rate of milk secretion and ultimately the milk yield is regulated by the rate of milk removal and, in part, this effect has been attributed to the increased rate of removal of an autocrine inhibitor in the more frequently milked gland (Wilde and Peaker, 1990). An additional effect of frequent milking is that mammary prolactin receptors are increased in the more frequently milked gland (McKinnon et aL, 1988) raising the possibility that the milking frequency may affect the sensitivity of the mammary gland to the endocrine environment. More recently, a whey fraction from goat milk has been shown to reduce the number of cell surface receptors for prolactin within 2 hr of addition to freshly prepared lactating mouse mammary cells (Bennett et al., 1990). From in vitro studies of mid-pregnant mammary gland explants, prolactin has been shown to be a major determinant of the induction of the lipogenic pathways in both rodents (Speake et al., 1975; Martyn and Falconer, 1985) and ruminants (Forsyth and Turvey, 1984; Barber et al., 1991). Although prolactin may play a less important role than growth hormone in ruminant lactation, the ability of the mammary gland to respond to circulating prolactin may be an important factor in the modulation of ACC and FAS gene expression by milking frequency. Acknowledgements--The research was funded by the Scottish Office Agriculture and Fisheries Department. We thank Dr D. Hirst for statistical advice and to Drs C. J. Wilde and C. H. Knight for useful discussions and for tissue samples. We thank Professor H. Rochefort for the gift of the FAS eDNA probe. REFERENCES
Aviv H. and Leder P. (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. natn. Acad. Sci. U.S.A. 69, 1408-1412. Bai D. H., Pape M. E., Lopez-Casillas F., Luo X., Dixon J. E. and Kim K. H. (1986) Molecular cloning of eDNA for acetyl-CoA.enzymeA carboxylase. J. bioL Chem. 261, 12395-12399. Barber M. C., Finley E. and Vernon R. G. (1991) Mechanisms whereby prolactin modulates lipogenesis in sheep mammary gland. Horm. Metab. Res. 23, 143-145. CBPB 103/1--!
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Barber M. C., Travers M. T., Finley E., Flint D. J. and Vernon R. G. (1992) Growth hormone-prolactin interactions in the regulation of mammary and adipose tissue acetyl~:~oA carboxylase activity and gene expression in lactating rats. Biochem. J. 285, 469-475. Bennett C. N., Knight C. H. and Wilde Co J. (1990) Regulation of mammary prolactin binding by secreted milk proteins. J. Endocr. 127, (suppl. 141). Chalbos D., Chambon M., Ailhaud G. and Rochefort H. (1987) Fatty acid synthetase and its mRNA are induced by progestins in breast cancer cells. J. biol. Chem. 262, 9923-9926. Chirgwin J. M., Przybyla A. E., MacDonald R. J. and Rutter W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 15, 5294-5299. Davis L. G., Dibner M. D. and Battey J. F. (1986) Basic Methods in Molecular Biology (Edited by Davis L. G., Dibner M. D. and Battey J. F.) Elsevier, Amsterdam. Erlich H. A., Gelfand D. and Sninsky J. J. (1991) Recent advances in the polymerase chain reaction. Science 252, 1643-1650. Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Analyt. Biochem. 132, 6-13. Forsyth I. A. and Turvey A. (1984) Fatty acid synthesis by explant cultures from the mammary glands of goats on days 60 and 120 of pregnancy. J. Endocr. 100, 87-92. Frohman M. A. (1990) RACE: rapid amplification of eDNA ends. In PCR Protocols (Edited by Innis M. A., Gelfand D. H., Sninsky J. J. and White T. J.), pp. 28-38. Academic Press, New York. Henderson A. J., Blatchford D. R. and Peaker M. (1983) The effects of milking thrice, instead of twice, daily on milk secretion in the goat. Q. J. exp. Physiol. 68, 645~52. Henderson A. J., Blatchford D. R. and Peaker M. (1985) The effects of long-term thrice-daily milking on milk secretion in the goat: evidence for mammary growth. Q. J. exp. Physiol. 70, 557-565. Innis M. A., Myambo K. B., Gelfand D. H. and Brow M. A. D. (1988) DNA sequencingwith Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA. Proc. hath. Acad. Sci. U.S.A. 85, 9436-9440. Kim K. H., Lopez-CasillasF., Bai D. H., Luo X. and Pape M. E. (1989) Role of reversible phosphorylation of acetyl~2oA carboxylase in long-chain fatty acid synthesis. FASEB J. 3, 2250-2256. Knight C. H., Fowler P. A. and Wilde C. J. (1990) Mammogenic effects of long-term treatment with bovine somatotrophin (BST) and thrice-daily milking in goats. J. Endocr. 127, 129-138. Lopez-Casillas F., Bai D. H., Luo X., Kong I. S., Hermodson M. A. and Kim K. H. (1988) Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase. Proc. hath. Acad. Sci. U.S.A. 85, 5784-5788. Majerus P. W. and Kilburn E. J. (1969) Acetyl-CoA carboxylase: the roles of synthesis and degradation in regulation of enzyme levelsin rat liver. J. biol. Chem. 244, 6254-6262. Martyn P. and Falconer I. R. (1985) The effect of progesterone on prolactin stimulation of fatty acid synthesis, giyeerolipid synthesis and lipogenic enzyme activities in mammary glands of pseudopregnant rabbits, after explant culture or intraductal injection. Biochem. J. 231, 321-328. McKinnon J., Knight C. H., Flint D. J. and Wilde C. J. (1988) Effect of milking frequency and efficiencyon goat mammary prolactin receptor number. J. Endocr. 119, (suppl. 167). Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K. and Green M. R. (1984) Efficient in vitro
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synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035-7056. Russel M., Kidd S. and Kelly M. S. (1986) An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene 45, 333-338. Sanger F., Nicklen S. and Coulson A. R. (1977) DNA sequencing with chain terminating inhibitors. Proc. natn. Acad. Sci. U.S.A. 74, 5463-5467. Speake B. K., Dils R. and Mayer R. J. (1975) Regulation ofenzyme turnover during tissue differentiation. Biochem. J. 148, 309-320. Takai T., Yokoyama C., Wada K. and Tanabe T. (1988)
Primary structure of chicken liver acetyl-CoA earboxylase deduced from eDNA sequence. J. bioL Chem. 263, 2651-2657. Wilde C. J. and Peaker M. (1990) Autoerine control in milk secretion. J. Agric. Sci. 114, 235-238. Wilde C. J., Henderson A. J., Knight C. H., Blatehford D. R., Faulkner A. and Vernon R. G. (1987) Effect of long-term thrice-daily milking on mammary enzyme activity, cell population and milk yield in the goat. J. Anita. Sci. 64, 533-539. Wilde C. J., Blatchford D. R. and Peaker M. (1991) Regulation of mouse mammary cell differentiation by extracellular milk proteins. Expl PhysioL 76, 379-387.
0305-0491/93$6.00+0.00 © 1993PergamonPress Ltd
ISOLATION OF A GOAT ACETYL-COA CARBOXYLASE COMPLEMENTARY D N A A N D EFFECT OF MILKING FREQUENCY ON THE EXPRESSION OF THE ACETYL-COA CARBOXYLASE AND FATTY ACID SYNTHASE GENES IN GOAT MAMMARY GLAND MAUREEN T. TRAVERS and MICHAEL C. BARBER*
Hannah Research Institute, Ayr, KA6 5HL, Scotland, U.K. (Tel. 0292-76013; Fax 0292-671052) (Received 28 August 1992; accepted 11 November 1992)
Abstract--1. Using the polymerase chain reaction we have isolated a partial complementary DNA for goat acetyl-CoA carboxylase which is 90 and 82% homologous to the published rat and chicken complementary DNA sequences, respectively. 2. Frequent milking causes an upregulation of the acetyl-CoA carboxylase and fatty acid synthase genes in goat mammary gland that parallels the increase in the respective enzyme activities. 3. The sequence for goat acetyl-CoA carboxylase is in the EMBL data base, Accession Number Z17803.
INTRODUCTION
It is well established that milking dairy animals more frequently than the usual twice a day increases milk yield. This effect in the goat is both acute (within hours; Henderson et al., 1983) and chronic (after weeks; Henderson et al., 1985) and can be accounted for by immediate increases in the secretory activity of the mammary cells and by more long-term adaptations resulting initially in increased cellular activity followed by increases in the mammary cell population (Wilde et al., 1987). Several studies (Wilde et al., 1987; Knight et al., 1990) have shown that a consequence of long-term frequent milking is a cellular up-regulation of a number of enzymes of the lipogenie pathway, notably acetyl-CoA carboxylase (ACC EC 6.4.1.2) and fatty acid synthase (FAS EC 2.3.1.85). The mechanism of this up-regulation is unknown though it is thought to relate, in part, to an increased rate of removal of an inhibitory autocrine factor in the more frequently milked gland (Wilde and Peaker, 1990). Recently, a 10-30kDa whey fraction purified from goat milk has been shown to inhibit the induction of FAS protein in cultures of mid-pregnant mouse mammary cells cultured on floating collagen gels (Wilde et al., 1991). In this present study we describe the cloning of a complementary DNA (eDNA) for goat ACC and show, using this eDNA together with a human FAS eDNA (Chalbos et al., 1987), that increased milking frequency results in increases in ACC and FAS gene
*To whom correspondence should be addressed.
Abbreviations--PCR: Polymerase chain reaction; bGH: recombinant bovine growth hormone; RNase: ribonuelease.
expressions that parallel the cellular increases in the enzyme activities. This suggests that the relief from chemical feedback inhibition in the more frequently milked gland results in increased ACC and FAS enzyme activity at least partly through a pre-translational mechanism, possibly through increases in the transcription rate or stabilization of the messenger RNAs (mRNA), or both. MATERIALS AND METHODS
Animals
British Saanen goats from the Hannah Research Institute herd were used in their first-to-third lactation, as described previously (Knight et al., 1990). Ten goats at approximately the same stage of lactation and of similar body weight (70.6 + 1.1 kg, S E M ) were paired for milk yield (overall mean of 23.2 + 1.48 kg/week). At week 19 of lactation, one goat of each pair was injected daily with 0.15 mg recombinant bovine growth hormone (bGH)/kg and the other goat received the vehicle only, as described previously (Knight et al., 1990). Starting at the same time as the daily injections the right mammary gland of each goat was switched from twice- to thrice-daily milking (07.30, 15.30 and 23.30hr); the left gland remained on twice-daily milking (0.730 and 15.30hr). The experiment continued for 22 weeks at the end of which time the goats were killed and samples of mammary tissue were quickly excised and frozen in liquid nitrogen. Cloning o f a goat acetyl-CoA carboxylase eDNA by the polymerase chain reaction
Poly (A ÷ ) RNA was prepared from total cellular RNA (Chirgwin et al., 1979) using oligo (dT) 123
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MAUREENT. TRAVERSand M1cr~.~LC. BARBER
cellulose chromatography (Aviv and Leder, 1972). For the preparation of first strand eDNA, 1/~g of poly (A + ) RNA was denatured and then annealed to 5 #g random hexamers (Pharmacia, Uppsala, Sweden). The final reaction conditions were 50/~g/ml poly (A +) RNA, 250#g/ml random hexamers, 50raM Tris-HC1, pH8.3, 50mM KC1, 10mM MgC! 2, 1 mM EDTA, 10/~g/ml BSA, 1 mM DTT, 0.5 mM spermidine, 1.5 mM of each deoxynucleoside triphosphate, 1000U/ml RNAguard (Pharmacia), 150/~Ci/ml~ [32p]dCTP (3000 Ci/mmol, Amersham, Bucks, U.K.) and 250U/ml avian myeloblastosis virus reverse transcriptase (Pharmacia) in a total volume of 20 #1. The reaction was incubated at 42°C for 90 min. EDTA and NaOH were added to give a final concentration of 20 mM and 0.2 M respectively and the mixture incubated at 60°C for 1 hr. The eDNA was precipitated by addition of 0.1 vol 3M sodium acetate, pH 6.0, and 2 vol absolute ethanol. The efficiency of the reaction was determined by the incorporation of the [32p] dCTP into the eDNA and was generally in the range 25-30% of input RNA. To amplify a cDNA sequence corresponding to goat acetyl-CoA carboxylase 50 ng first strand eDNA was added to 100#1 of Polymerase Chain Reaction (PCR) buffer (10% v/v dimethyl sulphoxide, 67mM TrisHCI, pH 8.8, 7.6mM MgC12, 16.6mM (NH4)2SO4, 1 mM 2-mercaptoethanol, 67/~M EDTA, 170/~g/ml bovine serum albumin and 1.5mM each deoxynucleoside trophosphate (Frohman, 1990) containing 100 pmol each of the oligodeoxynucleotide upstream and downstream primers. The oligodeoxynucleotides were synthesized on an Applied Biosystems 81A DNA synthesizer. The upstream primer (5'GAAAACCCAGATGAGGGGTTTAAGCCCAG 3') and downstream primer (5' AGTCATTACCATCTTCATTACCTCAATCTCTGCATA 3') were complementary to the 3' ends of the double-stranded eDNA sequence encompassing amino acid residues 512-789 of the coding region of rat mammary gland acetyl-CoA carboxylase (Lopez-Casillas et al., 1988). The mixture was denatured at 95°C for 5 min and after cooling to 72°C, 2.5 units Thermus aquaticus (Taq) DNA polymerase (Anglian Biotec. Ltd, Colchester, UK) were added. The reaction mixture was then subjected to 45 cycles of denaturing at 92°C for 30 sec, annealing of the primers at 40°C for 30 sec and extending the eDNA at 72°C for 1 min. The resulting PCR product was phenol/chloroform extracted, the 3' ends flushed with T4 DNA polymerase (Davis et al., 1986) and cloned into the pGEM 3zf (___) (Promega) series of phagemid vectors. Singlestranded DNA was produced from the pGEM 3zf ( + ) phagemids by infection of recombinant JM 109 E. coli with R408 Helper phage (Russel et al., 1986). Both strands of the cloned PCR product was sequenced using the dideoxy chain termination method (Sanger et al., 1977) with the aid of sequencing grade Taq DNA polymerase (Promega) (Innis et al., 1988).
Northern blot analysis o f R N A and enzyme measurements
Forty micrograms of total cellular RNA was separated by electrophoresis in 1.2% agarose gels containing 2.2 M formaldehyde and then transferred to hybond N nylon membranes (Amersham) (Davis et al., 1986). For the measurement of FAS mRNA abundance we obtained a human FAS eDNA (H. Rochefort, INSERM, France; Chalbos et al., 1987). The human FAS eDNA insert was sub-cloned into pGEM 3zf ( + ) and a labelled antisense RNA probe was generated from the SP6 promoter (Melton et al., 1984). For the measurement of ACC mRNA the cloned ACC PCR product was labelled to high specific activity by random priming (Feinberg and Vogelstein, 1983). Membranes were hybridized at 42°C (ACC eDNA probe) or 55°C (FAS riboprobe) overnight in hybridization solution (50% formamide, 0.75 M NaCI, 50 mM sodium dihydrogen phosphate, pH 7.4, 5 mM EDTA and Ficoll 400, bovine serum albumin, polyvinylpyrolidone and SDS each at 0.1% (w/v), and 200/~g of denatured salmon testes DNA/ml containing labelled probe at 2 x 10 6 dpm/ ml. After hybridization the membranes were washed in 15mM NaCI, 1.5mM sodium citrate, 0.1% w/v SDS at 50-55°C. An RNase digestion step was included before the final wash of membranes hybridized with the antisense FAS RNA probe. Membranes were exposed to XAR-5 film at - 7 0 ° C with intensifying screens. The autoradiographs were scanned using a Biorad laser densitometer with 1-D software. Total ACC and FAS enzyme activities were collated from previously published data (Knight et al., 1990). Statistical analysis
Differences were assessed by analysis of variance fitting the effects of twice- vs thrice-daily milking alone, the effect of bGH alone and the interaction between bGH and differential milking. As the effects o f b G H or the interaction between bGH and differential milking never approached significance (P in the range 0.75-0.90) data from bGH-treated goats and control goats was pooled and the results have been expressed in terms of the effects of twice- vs thricedaily milking. RESULTS
Amongst the variety of purposes that PCR has been applied to is the cross-species isolation of homologous genes (Erlich et al., 1991). A common feature of most applications of PCR is the use of primers designed to match two known or presumed eDNA or genomic DNA sequences. Previous to this present study the eDNA sequences for chicken (Takai et al., 1988) and rat (Lopez-Casillas et al., 1988) acetylCoA carboxylase have been reported. Comparison of the coding region of the two acetyl-CoA carboxylase cDNAs shows them to be 80 and 96% homologous
125
Mammary gland lipogenic enzyme gene expression in eDNA sequence and amino acid sequence, respectively, in a region 1 kb upstream of the biotin binding site. In this present study, by applying a strategy of using relatively long primers derived from the rat ACC eDNA that show greater-than-average sequence homology with the homologous chicken ACC eDNA, we have been able to amplify an 834bp cDNA from lactating goat mammary gland eDNA. Because the downstream primer corresponds to the region coding for the biotin-binding site of the protein the 834 bp cDNA encompasses 768 bp of novel sequence just upstream of the putative biotin-binding site and is of the predicted size by comparison with
GOAT RAT CHICKEN
GOAT RAT CHICKEN
GOAT RAT CHICKEN
GOAT RAT CHICKEN
GOAT RAT CHICKEN
GOAT RAT CHICKEN
GOAT RAT CHICKEN
GOAT RAT CHICKEN
GOAT RAT CHICKEN
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the homologous rat and chicken cDNAs. DNA sequence analysis of this goat ACC eDNA, which corresponds to about 11% of the eDNA encompassing the putative coding region, shows it to be 90 and 82°/. homologous to the corresponding rat and chicken eDNAs, respectively; the putative amino acid sequence is 96.1 and 94.5% homologous, respectively (Fig. 1). Amplification of rat mammary gland eDNA with the two PCR primers yielded a sequence 99.6% homologous to the published rat ACC sequence (Lopez-Casillas et aL, 1988) and use of this rat ACC eDNA in Northern analysis of rat mammary gland and adipose tissue RNA has shown a marked
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Fig. 1. Partial nucleotide sequence of goat acetyl-CoA carboxylase obtained from PCR amplification. The deduced amino acid sequence for goat acetyl-CoA carboxylase is displayed below the nucleotide sequence in one letter code together with the previously published amino acid sequences for rat (Lopez-Casillas et aL, 1988) and chicken (Takai et aL, 1988) acetyl-CoA carboxylase for comparison. The numbers correspond to the amino acid notation in rat acetyl-CoA carboxylase (Lopez-Casillas et aL, 1988).
126
MAUREEN T. TRAVERS and MICHAEL C. BARBER
A
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Fig. 2. Northern blot analysis of 40 #g total cellular RNA from a lactating goat milked three times (3 × ) a day on one mammary gland and twice a day (2 x ) on the other for 22 weeks. (A) Hybridization with goat ACC cDNA probe. (B) Hybridization with the human FAS riboprobe. The 11.5 kb ACC mRNA and the 10.7 kb FAS mRNA were integrated by densitometry (data summarized in Table 1).
concordance with ACC enzyme activities, suggesting transcriptional control in these rat tissues (Barber et al., 1992). Northern blot analysis (Fig. 2A) showed hybridization of the ACC cDNA probe to a major mRNA species of 11.5 kb in lactating goat mammary tissue, which is similar in size to the reported rat ACC mRNA (Bai et aL, 1986). A minor hybridizing species of around 3.8 kb is also observed with the goat ACC cDNA probe. This species has also been observed in RNA from rat tissues and has been attributed to degraded ACC mRNA (Bai et al., 1986). In lactating rats subjected to a variety of endocrinological manipulations, the abundance of the major ACC mRNA and of the 3.8 kb species in mammary and adipose tissue RNA varied in parallel with changes in total A C C enzyme activity, giving some support to this assertion (Barber et al., 1992). The 11.5 kb species represents the most functionally active species and therefore, in this present study, this has been primarily considered in the comparisons that have been made between total enzyme activity and functional acety142oA carboxylase gene expression. Conversely, the human FAS riboprobe hybridized to an mRNA species of approximately 10kb (Fig. 2B) which is slightly larger than the reported size for the human mRNA of 8 kb (Chalbos et al., 1987). Use of the ACC cDNA and FAS riboprobe in experiments to assess the effects of differential milking on ACC and FAS gene expression in lactating goat mammary gland showed that prolonged thricedaily milking produced a marked increase (P <0.01) in the amount of ACC and FAS mRNA/mg DNA compared with twice daily milking and this paralleled the increases observed for ACC and FAS enzyme activity/mg DNA (Table 1). This effect appears to be
achieved in two ways in that thrice-daily milking not only increases (P < 0.05) the abundances of ACC and FAS mRNAs per unit total cellular RNA (Fig. 2; Table 1) but also results in a small but significant (P <0.05) increase in the amount of RNA per cell (Table 1; Wilde et al., 1987). This suggests that the effect of thrice-daily milking to overcome chemical feedback inhibition is both specific in terms of its effect on ACC and FAS gene expression but also more general in terms of the increase of the RNA content per mammary cell. DISCUSSION
The present study extends the observations of Knight et al., (1990) to show that the increase in total Table 1. Effect of milkingfrequencyon ACC and FAS gene expression and enzymeactivitiesin goat mammarygland Milkingfrequency 2x 3× mg RNA/mg D N A
ACC m R N A abundance U/40 # g R N A FAS m R N A abundance U/40 # g R N A Units ACC mRNA/mg DNA Units FAS mRNA/mg DNA Enzyme activity~" ACC (nmol/mio/mg DNA) FAS (nmol/min/mg DNA)
0.77 _+ 0.09
1.00 _+ 0.08*
0.73 _+_0.24
1.74 + 0.52*
2.57 _+ 0.48
4.54 _+ 0.88*
16.6 _+ 6.5
41.0 _+ 3.2**
53.4 _+ 14.3
114.0 _+ 23.0**
73.7 _+ 8.9 453 + 85
97.0 _+ 6.7** 774 _+ 75**
Total cellular RNA was isolated from goat mammaryglands differentiallymilkedfor 22 w~eks.AcetyI-CoAcarboxylase and fatty acid synthasemRNA abundancewas determined by Northern analysisand has been express~ as the integrated area of the 11.5kb ACCmRNAand the 10.7kb FAS mRNAnormalizedagainst28s ribosomalRNA. Each point is the mean +_ SEM of 10 animals.
*P<0.05, **P<0.01 compared with 2 x gland. ~'Data from Knight et al., (1990).
Mammary gland lipogenic enzyme gene expression ACC and FAS enzyme activities associated with thrice-daily milking is caused in part by an increase in the abundances of the mRNAs for ACC and FAS. However, these observations do not preclude the importance of other mechanisms such as the control of mRNA translation and enzyme degradation (Majerus and Kilburn, 1969) and control of enzyme activity by reversible phosphorylation (Kim et al., 1989) which undoubtedly play a role in the lipogenic potential of the tissue. Though the increase in the abundances of the ACC and FAS mRNAs may be due to an increase in the transcription rate or an increase in mRNA stabilization, or a combination of both, the means by which the effect overall is achieved is a matter of conjecture. Previous studies have shown that the rate of milk secretion and ultimately the milk yield is regulated by the rate of milk removal and, in part, this effect has been attributed to the increased rate of removal of an autocrine inhibitor in the more frequently milked gland (Wilde and Peaker, 1990). An additional effect of frequent milking is that mammary prolactin receptors are increased in the more frequently milked gland (McKinnon et aL, 1988) raising the possibility that the milking frequency may affect the sensitivity of the mammary gland to the endocrine environment. More recently, a whey fraction from goat milk has been shown to reduce the number of cell surface receptors for prolactin within 2 hr of addition to freshly prepared lactating mouse mammary cells (Bennett et al., 1990). From in vitro studies of mid-pregnant mammary gland explants, prolactin has been shown to be a major determinant of the induction of the lipogenic pathways in both rodents (Speake et al., 1975; Martyn and Falconer, 1985) and ruminants (Forsyth and Turvey, 1984; Barber et al., 1991). Although prolactin may play a less important role than growth hormone in ruminant lactation, the ability of the mammary gland to respond to circulating prolactin may be an important factor in the modulation of ACC and FAS gene expression by milking frequency. Acknowledgements--The research was funded by the Scottish Office Agriculture and Fisheries Department. We thank Dr D. Hirst for statistical advice and to Drs C. J. Wilde and C. H. Knight for useful discussions and for tissue samples. We thank Professor H. Rochefort for the gift of the FAS eDNA probe. REFERENCES
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Barber M. C., Travers M. T., Finley E., Flint D. J. and Vernon R. G. (1992) Growth hormone-prolactin interactions in the regulation of mammary and adipose tissue acetyl~:~oA carboxylase activity and gene expression in lactating rats. Biochem. J. 285, 469-475. Bennett C. N., Knight C. H. and Wilde Co J. (1990) Regulation of mammary prolactin binding by secreted milk proteins. J. Endocr. 127, (suppl. 141). Chalbos D., Chambon M., Ailhaud G. and Rochefort H. (1987) Fatty acid synthetase and its mRNA are induced by progestins in breast cancer cells. J. biol. Chem. 262, 9923-9926. Chirgwin J. M., Przybyla A. E., MacDonald R. J. and Rutter W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 15, 5294-5299. Davis L. G., Dibner M. D. and Battey J. F. (1986) Basic Methods in Molecular Biology (Edited by Davis L. G., Dibner M. D. and Battey J. F.) Elsevier, Amsterdam. Erlich H. A., Gelfand D. and Sninsky J. J. (1991) Recent advances in the polymerase chain reaction. Science 252, 1643-1650. Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Analyt. Biochem. 132, 6-13. Forsyth I. A. and Turvey A. (1984) Fatty acid synthesis by explant cultures from the mammary glands of goats on days 60 and 120 of pregnancy. J. Endocr. 100, 87-92. Frohman M. A. (1990) RACE: rapid amplification of eDNA ends. In PCR Protocols (Edited by Innis M. A., Gelfand D. H., Sninsky J. J. and White T. J.), pp. 28-38. Academic Press, New York. Henderson A. J., Blatchford D. R. and Peaker M. (1983) The effects of milking thrice, instead of twice, daily on milk secretion in the goat. Q. J. exp. Physiol. 68, 645~52. Henderson A. J., Blatchford D. R. and Peaker M. (1985) The effects of long-term thrice-daily milking on milk secretion in the goat: evidence for mammary growth. Q. J. exp. Physiol. 70, 557-565. Innis M. A., Myambo K. B., Gelfand D. H. and Brow M. A. D. (1988) DNA sequencingwith Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction amplified DNA. Proc. hath. Acad. Sci. U.S.A. 85, 9436-9440. Kim K. H., Lopez-CasillasF., Bai D. H., Luo X. and Pape M. E. (1989) Role of reversible phosphorylation of acetyl~2oA carboxylase in long-chain fatty acid synthesis. FASEB J. 3, 2250-2256. Knight C. H., Fowler P. A. and Wilde C. J. (1990) Mammogenic effects of long-term treatment with bovine somatotrophin (BST) and thrice-daily milking in goats. J. Endocr. 127, 129-138. Lopez-Casillas F., Bai D. H., Luo X., Kong I. S., Hermodson M. A. and Kim K. H. (1988) Structure of the coding sequence and primary amino acid sequence of acetyl-coenzyme A carboxylase. Proc. hath. Acad. Sci. U.S.A. 85, 5784-5788. Majerus P. W. and Kilburn E. J. (1969) Acetyl-CoA carboxylase: the roles of synthesis and degradation in regulation of enzyme levelsin rat liver. J. biol. Chem. 244, 6254-6262. Martyn P. and Falconer I. R. (1985) The effect of progesterone on prolactin stimulation of fatty acid synthesis, giyeerolipid synthesis and lipogenic enzyme activities in mammary glands of pseudopregnant rabbits, after explant culture or intraductal injection. Biochem. J. 231, 321-328. McKinnon J., Knight C. H., Flint D. J. and Wilde C. J. (1988) Effect of milking frequency and efficiencyon goat mammary prolactin receptor number. J. Endocr. 119, (suppl. 167). Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K. and Green M. R. (1984) Efficient in vitro
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