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Sexually dimorphic expression of a broad array of liver proteins involved in reproduction and xenobiotic metabolism is induced at puberty by sex-specific growth hormone patterns. An additional control of sex-dependent gene expression is conferred by Regulator of sex-limitation (Rsl) alleles. In variant rsl mice, females inappropriately express the male Sex-limited protein, Slp. We recently showed that a panel of male-specific liver genes is repressed by Rsl, accentuating sex differences in a hormone-independent manner. Here we map rsl to a region on Chromosome 13 comprised exclusively of KRAB (Kruppel-associated box) zinc-finger protein (ZFP) genes. Among eight Rsl candidate (Rslcan) genes within the critical genetic interval, the recent duplicates Rslcan-4 and Rslcan-9 both harbor mutations in rsl mice (partial deletion and splice-site inactivation, respectively). Transgenesis with bacterial artificial chromosome (BAC) clones encompassing Rslcan-4 restores male-specific MUP (major urinary protein) expression to rsl mice, whereas a BAC containing Rslcan-9 rescues sex-specific expression of Slp and cytochrome P450 Cyp2d9. Thus, the Rslcan-4 and Rslcan-9 paralogs partitioned regulation of their target genes during evolution. This demonstrates the first biological role for a set of KRAB zinc-finger repressor proteins and reveals the molecular basis of a gene-silencing pathway critical for sexual dimorphism.

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, usingd-[3-3H, U-14C]glucose as the precursor of the lipid glycerol backbone shows that about 40–50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.

In the accompanying paper (James, P. F., and Zoeller, R. A. (1997) J. Biol. Chem. 272, 23532-23539), we reported the isolation of a series of mutants from the fibroblast-like cell line, CHO-K1, that are deficient in the incorporation of the long chain fatty alcohol, hexadecanol, into complex lipids. All but one of these mutants, FAA. K1B, were deficient in long-chain-fatty alcohol oxidase (FAO) activity. We have further characterized this FAO+ isolate. FAA.K1B cells displayed a 40% decrease in [9,10-3H]hexadecanol uptake when compared with the parent strain. Although incorporation of hexadecanol into the phospholipid fraction was decreased by 52%, the cells accumulated label in alkylglycerol (20-fold over wild type). The increase in 1-alkylglycerol labeling corresponded to a 4-fold increase in alkylglycerol mass. Short term labeling with 32Pi showed a 45-50% decrease in overall phospholipid biosynthesis in FAA.K1B. Both diacyl- and ether-linked species were affected, suggesting a general defect in phospholipid biosynthesis. Mutant cells were able to partially compensate for the decreased biosynthesis by decreasing the turnover of the phospholipid pools. The primary lesion in FAA. K1B was identified as a 95% reduction in acyl/alkyl-dihydroxyacetone-phosphate reductase activity. Whole cell homogenates from FAA.K1B were unable to reduce either acyl-dihydroxyacetone phosphate (DHAP) or alkyl-DHAP, supporting the notion that the reduction of these two compounds is catalyzed by a single enzyme. These data suggest that the biosynthesis of diacyl phospholipids, in Chinese hamster ovary cells, begins with the acylation of dihydroxyacetone phosphate as well as glycero-3-phosphate and that the "DHAP pathway" contributes significantly to diacyl glycerolipid biosynthesis. Also, the severe reduction in acyl/alkyl-DHAP reductase activity in FAA.K1B resulted in only a moderate decrease in ether lipid biosynthesis. These latter data together with the observed increase in alkylglycerol levels support the existence of a shunt pathway that is able to partially bypass the enzymatic lesion.

Upon differential centrifugation of guinea pig intestine mucosal cells homogenate, fatty acyl-CoA:NADPH oxidoreductase (long chain alcohol forming) was found to be enriched in the light mitochondrial (L) fraction (sedimenting between 66,000 x g min and 500,000 x g min) which contained mainly mitochondria, lysosomes, and peroxisomes. Peroxisomes (marker enzymes: catalase and dihydroxyacetone phosphate acyltransferase) present in the L fraction were separated from other organelles in a Nycodenz density gradient centrifugation employing a vertical rotor. By comparing the distribution of acyl-CoA reductase with different marker enzymes in the gradient, it was concluded that this reductase is primarily localized in the microperoxisomes (microbodies). The topography of the membrane-bound enzyme in the isolated organelles was studied by checking its lability toward trypsin in the absence and presence of the detergent Triton X-100. The results suggested that acyl-CoA reductase is localized on the outer surface (cytosolic side) of microperoxisomal membrane.

The genomic locus on mouse chromosome 13 called Regulator of sex-limitation (Rsl) accentuates sex differences in hepatic gene expression. Females homozygous for variant rsl alleles express some otherwise male-specific liver proteins, such as sex-limited protein (Slp), major urinary proteins (MUPs), and members of the cytochrome P450 (cyp) 2d subfamily. We recently identified mutations in two genes, Rsl1 and Rsl2, accounting for the rsl phenotype. These genes encode KRAB zinc-finger proteins (KRAB-ZFPs) and are embedded within a cluster of over 20 similar genes. Mammalian genomes contain over 200 KRAB-ZFP genes, which act biochemically to repress transcription, but the Rsl genes are the first to have their biological functions elucidated. Here we compare Rsl1 and Rsl2 with their neighboring genes, tracing a series of duplication, inversion, and gene conversion events that have created subfamilies within the locus. Polymorphisms among inbred mouse strains and feral species suggest that mutations responsible for the rsl phenotype arose during the creation of inbred strains. Comparisons among mouse, rat, and human sequences show that the Rsl genes, like members of certain other multigene families, have diversified in a species-specific manner. The targets of Rsl regulation also vary between species, occurring in gene families with functions in steroid and xenobiotic metabolism (Cyp2d), reproduction (MUPs), and immunity (Slp). This suggests that the Rsl locus in mouse, and comparable KRAB-ZFP genes in other mammals, may play a role in speciation via modulation of expression of genes influencing reproductive fitness or behavior.

We have developed a two-step selection protocol to generate a population of Chinese hamster ovary (CHO) cell variants that are plasmalogen-deficient, but contain intact, functional peroxisomes (plasmalogen-/peroxisome+). This involved sequential exposures of a mutagenized cell population to photodynamic damage by using two different pyrene-labelled sensors, 9-(1´-pyrene)nonanol and 12-(1´-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of which displayed a severe decrease in plasmalogen biosynthesis. Further characterization of one of the plasmalogen-deficient isolates, NRel-4, showed that it contained intact, functional peroxisomes. Whole-cell homogenates from NRel-4 displayed severely decreased dihydroxyacetone phosphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRel-4 and another, recently described, plasmalogen-deficient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Purdue and Zoeller (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4475–4480] were hypersensitive to singlet oxygen, supporting the notion of plasmalogens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRel-4 upon restoration of plasmalogen content by supplementation with a bypass compound, sn-1-hexadecylglycerol. NRel-4 and other plasmalogen-/peroxisome+ strains will allow us to examine further the role of ether lipids in cellular functions without complications associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.

The cDNA encoding rat brain cytosolic acyl-CoA thioester hydrolase (ACT) has been cloned and sequenced, and the primary structure of the enzyme has been deduced. A partial amino acid sequence (38 amino acids) of the enzyme was determined using the peptides generated after CNBr digestion of the purified enzyme. Primers synthesized on the basis of this information were used to isolate two cDNA clones, each encoding the full length of the enzyme. The nucleotide sequences of these clones contained an open reading frame encoding a 358-amino acid polypeptide with a calculated molecular mass of 39.7 kDa, similar to that determined for the purified enzyme (40.9 kDa). The deduced ACT sequence showed no homology to the known sequences of any other thioesterases nor to any other known protein sequence. However, there was a strong homology to a number of expressed sequence tag human brain cDNA clones. The identity of the ACT cDNA was confirmed by the expression of ACT activity in Escherichia coli. There was a 10-15-fold increase in ACT-specific activity in the bacterial extracts after induction with isopropyl thiogalactoside, and the properties of the expressed enzyme (fusion protein) were the same as those of the purified rat brain ACT. Northern blot analysis showed that a 1.65-kilobase ACT transcript was present in rat brain and testis but not in any other rat tissues examined. However, the ACT mRNA was induced in the liver of rats that were fed Wy-14,643, a peroxisome proliferator and inducer of rodent liver cytosolic acyl-CoA thioesterase. These results indicate that the induced rat liver ACT is homologous to the constitutive rat brain ACT.