Defining the Importance of Phosphatidylserine Synthase 2 in Mice

Phosphatidylserine synthase 1 (Pss1) and phosphatidylserine synthase 2 (Pss2) produce phosphatidylserine by exchanging serine for the head groups of other phospholipids. Pss1 and Pss2 are structurally similar (∼32% amino acid identity) but differ in their substrate specificities, with Pss1 using phosphatidylcholine for the serine exchange reaction and Pss2 using phosphatidylethanolamine. Whether Pss1 and Pss2 are both required for mammalian growth and development is not known, and no data exist on the relative contributions of the two enzymes to serine exchange activities in different tissues. To address those issues and also to define the cell type-specific expression of Pss2, we generated Pss2-deficient mice in which a β-galactosidase marker is expressed from Pss2 regulatory sequences. Histologic studies of Pss2-deficient mice revealed very high levels of β-galactosidase expression in Sertoli cells of the testis and high levels of expression in brown fat, neurons, and myometrium. The ability of testis extracts fromPss2-deficient mice to catalyze serine exchange was reduced by more than 95%; reductions of ∼90% were noted in the brain and liver. However, we found no perturbations in the phospholipid content of any of these tissues. As judged by Northern blots, the expression ofPss1 was not up-regulated in Pss2-deficient cells and tissues. Testis weight was reduced inPss2-deficient mice, and some of the male mice were infertile. We conclude that Pss2 is responsible for the majority of serine exchange activity in in vitro assays, but a deficiency in this enzyme does not cause perturbations in phospholipid content or severe developmental abnormalities. Phosphatidylserine synthase 1 (Pss1) and phosphatidylserine synthase 2 (Pss2) produce phosphatidylserine by exchanging serine for the head groups of other phospholipids. Pss1 and Pss2 are structurally similar (∼32% amino acid identity) but differ in their substrate specificities, with Pss1 using phosphatidylcholine for the serine exchange reaction and Pss2 using phosphatidylethanolamine. Whether Pss1 and Pss2 are both required for mammalian growth and development is not known, and no data exist on the relative contributions of the two enzymes to serine exchange activities in different tissues. To address those issues and also to define the cell type-specific expression of Pss2, we generated Pss2-deficient mice in which a β-galactosidase marker is expressed from Pss2 regulatory sequences. Histologic studies of Pss2-deficient mice revealed very high levels of β-galactosidase expression in Sertoli cells of the testis and high levels of expression in brown fat, neurons, and myometrium. The ability of testis extracts fromPss2-deficient mice to catalyze serine exchange was reduced by more than 95%; reductions of ∼90% were noted in the brain and liver. However, we found no perturbations in the phospholipid content of any of these tissues. As judged by Northern blots, the expression ofPss1 was not up-regulated in Pss2-deficient cells and tissues. Testis weight was reduced inPss2-deficient mice, and some of the male mice were infertile. We conclude that Pss2 is responsible for the majority of serine exchange activity in in vitro assays, but a deficiency in this enzyme does not cause perturbations in phospholipid content or severe developmental abnormalities. Phosphatidylserine is an aminophospholipid that constitutes 5–10% of mammalian membrane phospholipids (1Voelker D.R. Vance D.E. Vance J. Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier, Amsterdam1996: 391-393Google Scholar). In mammals, phosphatidylserine is synthesized by a pair of enzymes, phosphatidylserine synthase 1 (Pss1) 1The abbreviations used are: Pss1, phosphatidylserine synthase 1; Pss2, phosphatidylserine synthase 2; CHO, Chinese hamster ovary; FSH, follicle-stimulating hormone; PBS, phosphate-buffered saline; β-gal, β-galactosidase; X-gal, 5-bromo-4-chloro-3-indolyl-β-dgalactopyranoside 2According to the International Committee on Standardized Genetic Nomenclature for Mice, the official gene symbols for phosphatidylserine synthase 1 and phosphatidylserine synthase 2 arePtdss1 and Ptdss2, respectively. In this paper, we have used the names Pss1 and Pss2 because they are less cumbersome. and phosphatidylserine synthase 2 (Pss2) (2Kuge O. Nishijima M. Biochim. Biophys. Acta. 1997; 1348: 151-156Google Scholar, 3Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1991; 266: 24184-24189Google Scholar, 4Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Google Scholar), located primarily within the mitochondria-associated membrane fraction of the endoplasmic reticulum (5Vance J.E. J. Biol. Chem. 1991; 266: 89-97Google Scholar, 6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Google Scholar). The two enzymes are structurally related, with 32% amino acid identity, and both are predicted to contain several transmembrane domains (2Kuge O. Nishijima M. Biochim. Biophys. Acta. 1997; 1348: 151-156Google Scholar, 3Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1991; 266: 24184-24189Google Scholar, 6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Google Scholar, 7Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Google Scholar). Pss1 and Pss2 generate phosphatidylserine by catalyzing the exchange of serine for the head group of another phospholipid, but the two enzymes differ in their substrate specificities. Pss1 uses phosphatidylcholine for the exchange reaction (8Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Google Scholar, 9Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Google Scholar), whereas Pss2 uses phosphatidylethanolamine (2Kuge O. Nishijima M. Biochim. Biophys. Acta. 1997; 1348: 151-156Google Scholar, 3Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1991; 266: 24184-24189Google Scholar, 4Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Google Scholar, 6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Google Scholar, 10Saito K. Nishijima M. Kuge O. J. Biol. Chem. 1998; 273: 17199-17205Google Scholar). In vitro, Pss1 is capable of catalyzing the exchange of ethanolamine and choline in addition to serine; Pss2 is capable of catalyzing the exchange of ethanolamine but not choline. However, these ethanolamine and choline exchange reactions are not thought to be physiologically important for the in vivo synthesis of phosphatidylethanolamine (11Sundler R. Akesson B. Nilsson A. FEBS Lett. 1974; 43: 303-307Google Scholar) or phosphatidylcholine (12Sundler R. Arvidson G. Akesson B. Biochim. Biophys. Acta. 1972; 280: 559-568Google Scholar). The physiologic “rationale” for the existence of two different phosphatidylserine synthases is unclear. No one knows whether mammalian growth and development require both enzymes, since no one has yet developed mice lacking either of the two genes. However, two groups have produced Chinese hamster ovary (CHO) cell lines lackingPss1 by selecting for cells that required ethanolamine or phosphatidylserine for growth (8Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Google Scholar, 9Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Google Scholar). Extracts from the mutant CHO cells manifested a ∼50% decrease in serine exchange activity, suggesting that Pss2 accounts for a significant portion of the serine exchange activity in that cell type. Pss1 deficiency did not have a significant impact on cell growth when the cells were grown in the presence of ethanolamine or phospholipids, but the cells grew slowly, and their phospholipid content was perturbed when the cells were grown in the absence of ethanolamine, phosphatidylserine, or phosphatidylethanolamine (7Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Google Scholar, 8Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Google Scholar, 9Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Google Scholar, 13Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1999; 274: 23844-23849Google Scholar). Thus far, no one has developed cell lines that lack Pss2expression, although a mutant CHO cell line that expressed reduced levels of Pss2 activity was generated (10Saito K. Nishijima M. Kuge O. J. Biol. Chem. 1998; 273: 17199-17205Google Scholar). It is difficult to predict whether or not Pss2-deficient cells would be viable and healthy. On the one hand, one could argue that the existence ofPss1 would make Pss2 expression superfluous. On the other hand, a large fraction of phosphatidylserine synthesis in CHO cells is due to Pss2, and it certainly would not be unreasonable to surmise that Pss2 would be crucial for cellular phospholipid homeostasis. It is also difficult to make a prioripredictions about whether mice lacking Pss2 would be viable and, if so, whether there would be any pathology in tissues expressing high levels of the enzyme. Pss2 is expressed in a variety of different organs as judged by Northern blot analysis (6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Google Scholar, 7Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Google Scholar), but no information exists on which cell types express high levels of the gene. Finally, there are no biochemical data on the contribution of Pss2 to total serine exchange activity in different mammalian tissues. The purpose of this study was to define the physiologic importance ofPss2 in mammals. To address this issue, we producedPss2-deficient mice in which the expression of a marker gene, β-galactosidase (β-gal), was driven by the regulatory elements of the Pss2 gene. The characterization of thePss2-deficient mice allowed us to fill in a number of gaps in our knowledge of Pss2. First, we demonstrated that mice lacking Pss2 survive development and are viable, although males have small testes and occasionally testicular atrophy. We were able to define the impact of Pss2 deficiency on serine exchange activities in different tissues and were able to demonstrate, by β-gal staining, which cell types express high levels ofPss2. Finally, we investigated whether or notPss2 deficiency perturbed the phospholipid composition of tissues and cells. A mouse embryonic stem cell line (KST314, strain 129P2/OlaHsd) containing an insertional mutation in Pss2 was identified in a gene-trapping screen (14Mitchell K.J. Pinson K.I. Kelly O.G. Brennan J. Zupicich J. Scherz P. Leighton P.A. Goodrich L.V. Lu X. Avery B.J. Tate P. Dill K. Pangilinan E. Wakenight P. Tessier-Lavigne M. Skarnes W.C. Nat. Genet. 2001; 28: 241-249Google Scholar). The gene-trapping vector, pGTITMpfs, was designed to interrupt genes that encode proteins with an N-terminal signal sequence and to create an in-frame fusion with the β-geo reporter gene (14Mitchell K.J. Pinson K.I. Kelly O.G. Brennan J. Zupicich J. Scherz P. Leighton P.A. Goodrich L.V. Lu X. Avery B.J. Tate P. Dill K. Pangilinan E. Wakenight P. Tessier-Lavigne M. Skarnes W.C. Nat. Genet. 2001; 28: 241-249Google Scholar). The embryonic stem cell line was used to generate male chimeric mice, which were bred with C57BL/6 mice to establish heterozygous (Pss2+/−) and homozygous (Pss2−/−) knockout mice. Mice were genotyped by quantifying neomycin phosphotransferase II (neo) gene dosage in genomic DNA with a quantitative PCR assay (described on the BayGenomics Web site at baygenomics.ucsf.edu/protocols). Genotyping was also performed by quantifying neo gene dosage with Southern blots; for these studies, BamHI-digested genomic DNA was hybridized with pGTITMpfs that had been linearized with HindIII. All mice described here had a mixed genetic background (∼50% C57BL/6 and ∼50% 129/OlaHsd). The mice were weaned at 21 days of age, housed in a barrier facility with 12-h light/dark cycle, and fed a chow diet containing 4.5% fat (Ralston Purina, St. Louis, MO). Pss2−/−, Pss2+/−, and wild-type (Pss2+/+) embryonic fibroblasts were prepared from 13.5-day mouse embryos (15Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Google Scholar) and immortalized by serial passaging (15Kim E. Ambroziak P. Otto J.C. Taylor B. Ashby M. Shannon K. Casey P.J. Young S.G. J. Biol. Chem. 1999; 274: 8383-8390Google Scholar). Sites of Pss2 andPss1 expression were determined with mouse multiple-tissue poly(A)+ RNA blots (CLONTECH, Palo Alto, CA). The protein-coding sequence of the Pss1 cDNA was amplified from a mouse liver cDNA library (CLONTECH) with oligonucleotides 5′-ATGGCGTCCTGCGTGGGGAGCAGG-3′ and 5′-CAGCCGATGAAGAGGATTCTACACC-3′ and cloned into pCRII (Invitrogen). A 1.1-kb Pss1 cDNA probe was produced by removing the insert with EcoRI. A 1.4-kbPss2 cDNA was cloned into pCRII (7Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Google Scholar); the insert was released by EcoRI digestion. Probes were labeled with32P by random hexamer priming. The Northern blots were exposed to x-ray film for 12 h at −80 Northern blots were also produced with total RNA from and testis from and mice. RNA was with the RNA RNA was by on a and to a membrane and hybridized with the 1.4-kbPss2 cDNA The blots were also with the 1.1-kb Pss1 cDNA probe and a neo probe by with M. Scholar). were with a in a containing and The was for at and the was used for of serine exchange The reaction of with of of a and and of the an or a The of in a of and of serine The of in a of and of ethanolamine The of in a of and of choline The reaction was allowed to for at and was by of A total of of was to and the were at for The was and The was with of The phospholipid were by the of and J. Biochem. Scholar), and was fibroblasts from embryos were grown to in in the presence of The cells were for to h with were by of the cells with and by in the were by to and phosphatidylserine identified by to in phosphatidylserine was determined by of tissues from mice were on The were in phosphate-buffered with for at and with The were with an for h at The was prepared by 1 of a of in with of containing and 2 staining, the were with PBS, for 2 with and for with The were with and and with CA). from mice and and were from and was from mice and and and levels were with an 1972; Scholar). were from or fibroblasts by the of and J. Biochem. Scholar). The fraction was and with The was a of and were in containing composition was determined with a high with a to an The of as a to in by a for was used for data and data were for the different phospholipids by of of and testes were from and mice and in a in of containing and cells and were by for were with a for The from the was to for 1 h at the was as were from the J. Biochem. Scholar) and by in the were identified by to and with to phosphatidylserine, and were from the and the of phospholipid of was determined by of X. G. J. Scholar). Pss1 are both expressed in a and group of as judged by multiple-tissue Northern blots A and is expressed at high levels in the but Pss1 is also expressed Pss1 is expressed in liver and but Pss2 is also expressed in those tissues. To whether expression levels of both of the phosphatidylserine synthases are required for mammalian development and for of mice, we generated Pss2 knockout mice with an embryonic stem cell line containing an insertional mutation in 2 of Pss2. The mutation in the of a fusion containing Pss2 the amino of the enzyme the transmembrane to the transmembrane and from the gene fusion and G. P. 1991; Scholar, W.C. J.E. A. Scholar). The of the Pss2 fusion is the of the Pss2 regulatory sequences. mice and at the as mice. The and phospholipid levels were no different than in wild-type not mice as did the majority of the but of the male mice were or The absence of a severe was not due to of the insertional mutation of a Pss2 from the mutant No Pss2 was in knockout mice 1 As a fusion could be on a Northern blot hybridized with a neo probe 1 The knockout of Pss2 did not in either in fibroblasts from embryos 1 or in the testis or brain of mice 1 To the contribution of Pss2 to total phosphatidylserine synthase activity in mammalian we the ability of extracts to catalyze the exchange of ethanolamine, and choline into phospholipids. exchange activity was in extracts from testes than in extracts testes exchange was also reduced in the and liver mice, although to a than in the testes 2 The ability of extracts mice to exchange ethanolamine was also reduced 2 choline which is of Pss1 activity, was by about tissues 2 exchange activities in tissues from mice were those of and mice not Pss1 deficiency in CHO cells reduced phosphatidylserine synthase activity, and phospholipid composition of the mutant cells was perturbed when the cells were grown in or (6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Google Scholar, 8Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Google Scholar, 9Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Google Scholar, 13Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1999; 274: 23844-23849Google Scholar). To whether Pss2 deficiency phospholipid we the phospholipid content of the and testis of mice and No significant were We also the phospholipid content of from the liver and testis and mice. no significant were composition of from liver and testis and testis from mice mice were and were by are from mice and We the that the phospholipid composition of tissues have been due to an of phospholipids at the as a of the of phospholipids to tissues by To that we serine exchange activity and phospholipid content of and fibroblasts grown for h in exchange fibroblasts was reduced by ∼90% no significant in cellular phospholipid composition was noted To whether the reduced serine exchange activity in extracts from fibroblasts to a decrease in phosphatidylserine synthesis in we determined the of serine into Phosphatidylserine synthesis was reduced in fibroblasts The cell types responsible for high levels of Pss2 expression are To address this issue, we β-gal expression of Pss2 in mouse embryos and in mice. In the levels of β-gal expression were in brown the and in As the brown with In mice, β-gal was in the and In the β-gal expression was to and was in the cells of the and in in the of the levels of β-gal expression were also in the of the In testes of mice we two In the majority of the mice testis by with small of cells in the Sertoli cells the and and In the mice, the testes were of and revealed of cells and with a of Sertoli cells and no We testes in or of whether the mice was or β-gal of Sertoli cells was A and was in Sertoli cell to but no of or was revealed β-gal within but the was than in Sertoli cells. The of testis in of mice but in of the that Pss2 deficiency cause testis pathology but with this were the we that a more of mice in testis or in with testis To this we testis in mice with testis and in mice studies revealed a small but significant in testis weight in mice Sertoli cells produce which levels S.J. E. M. J. 2001; Scholar). Sertoli cell to levels and high levels S.J. E. M. J. 2001; Scholar). We that Sertoli cell in mice be by this was the levels in mice were than in the a of biochemical and genetic studies have that have two different enzymes for phosphatidylserine, Pss1 and Pss2 (2Kuge O. Nishijima M. Biochim. Biophys. Acta. 1997; 1348: 151-156Google Scholar, 3Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1991; 266: 24184-24189Google Scholar, 4Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Google Scholar, 7Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Google Scholar). two enzymes have significant sequence but differ in their substrate specificities. In this we mice and used those mice and cell lines to address a number of the mammalian studies have important information to the First, it that both enzymes are not required for The that homozygous Pss2-deficient mice are viable, and phospholipid is for at we by β-gal staining, the cell types that express Pss2 at high The levels of Pss2 expression were located in the brown fat development and in the Sertoli cells of the testis in mice. we demonstrated that Pss2 accounts for the majority of serine exchange activity in mammalian tissues. we demonstrated that Pss2 is not required for the of phosphatidylserine levels in when the cells are of ethanolamine and phospholipids. was noted in of male mice, and male mice lacking testicular had testes than We that these testes could to a to phosphatidylserine in Sertoli which to the cells within the Biol. Scholar, Biol. 1998; Scholar). from the reduced testis Sertoli cell was also by the we could not any significant perturbations in phospholipid composition in extracts of although serine exchange activity was reduced by It is that phosphatidylserine levels could be reduced in a of the cells in the as the Sertoli but we that any reductions would be and be within the in phospholipids. Extracts from Pss2-deficient tissues manifested an in choline exchange exchange is by Pss1 O. Saito K. Nishijima M. J. Biol. Chem. 1997; 272: 19133-19139Google Scholar, 6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Google Scholar, 8Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Google Scholar). A for the would have been an in Pss1 expression in to the deficiency in Pss2. However, as judged by Northern expression was both in mouse tissues and in fibroblasts We an that the of choline exchange in tissues a in Pss1 Kuge O. K. Saito K. Nishijima M. A. 1998; Scholar) demonstrated that Pss1 activity is reduced by levels of phosphatidylserine in and they identified a amino acid within Pss1 that is crucial for the of the enzyme. Pss2 deficiency could in levels of phosphatidylserine in some regulatory in the of Pss1 activity and the levels of choline exchange exchange activity was reduced by ∼90% inPss2-deficient but the phospholipid composition was when the fibroblasts were grown in and exchange activities were in and and there were no perturbations in phospholipids. The of these is that the levels of serine exchange activity in mice due to are to levels of phosphatidylserine and phosphatidylethanolamine. However, two be in phosphatidylserine and phosphatidylethanolamine levels are not inPss2-deficient tissues. First, the ∼90% decrease in serine exchange activity in Pss2-deficient tissues was with an in vitro activity and that of assay not the of phosphatidylserine synthesis in the The in vitro assay is performed for which the of enzyme is whereas in the cell other the of the presence of and the of the of phosphatidylserine although of serine exchange activity in extracts was by Pss2 we be that the of phosphatidylserine synthesis was reduced to a in cell and and more a to an of phospholipid synthesis is a in the of phospholipid (6Stone S.J. Cui Z. Vance J.E. J. Biol. Chem. 1998; 273: 7293-7302Google Scholar, 7Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Google Scholar, Biol. Scholar, Biol. 1998; Scholar). the of phosphatidylserine synthesis could in a of phosphatidylserine phosphatidylethanolamine the of phospholipid The in fibroblasts that the synthesis of phosphatidylserine is reduced in with the in serine exchange However, is in those The cell lines used for those were from different embryos were not on an and had In this one the that genetic from the Pss2 could have had an on phosphatidylserine synthesis In the we that it be to a analysis of phosphatidylserine and in cell lines and in tissues from Pss2-deficient mice. with CHO cells (8Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Google Scholar, 9Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Google Scholar). In the CHO serine exchange activity was reduced by ∼50% the but there were significant in phosphatidylserine and phosphatidylethanolamine levels when the cells were grown in and (7Stone S.J. Vance J.E. Biochem. J. 1999; 342: 57-64Google Scholar, 8Voelker D.R. Frazier J.L. J. Biol. Chem. 1986; 261: 1002-1008Google Scholar, 9Kuge O. Nishijima M. Akamatsu Y. J. Biol. Chem. 1986; 261: 5795-5798Google Scholar, 13Kuge O. Saito K. Nishijima M. J. Biol. Chem. 1999; 274: 23844-23849Google Scholar). is the of Pss1 deficiency in CHO cells We that the be by the different substrate of the two We deficiency is because Pss1 is capable of phosphatidylserine from when Pss2-deficient cells are of ethanolamine, phosphatidylserine to be produced from and phosphatidylethanolamine be generated by of In in the of Pss2 generate phosphatidylserine, but if phosphatidylethanolamine is for the exchange In the absence of ethanolamine, when phosphatidylethanolamine be generated from the there is no to significant of phosphatidylserine or phosphatidylethanolamine. In the of Pss1 phosphatidylethanolamine be generated from phosphatidylserine, and phosphatidylserine be generated from phosphatidylethanolamine. cells are on ethanolamine to their phosphatidylserine and phosphatidylethanolamine The of Pss2-deficient mice make it to address a of issues in the we are by the high levels of Pss2 expression in within the of phosphatidylserine are into and to phosphatidylethanolamine J.E. Scholar, S.J. Vance J.E. J. Biol. Chem. Scholar, M. G. R. J. Biol. Scholar, K. Kuge O. Nishijima M. M. A. 1999; Scholar). It would be to whether the of Pss2-deficient mice be more to in to that the of from or The studies also several other issues for on the is mice would to a deficiency in we would to whether Pss1 is for embryonic development of mammals, as was the for Pss2. mice we would to they would to a diet containing levels of We would also be in whether a knockout would mice. to these the We K. J. for the and characterization of the gene cell for and G. and E. Vance for on the

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