To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty acid synthase (FAS) gene in vivo, we generated lines of transgenic mice carrying 2.1 kilobases of the 5-flanking region (؊2100 to ؉67) of the rat FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. This reporter gene construct was strongly expressed in tissues that normally express high levels of FAS mRNA, which include liver and white adipose tissues. In contrast, CAT reporter activity was not detected in appreciable levels in lung, heart, kidney, and muscle tissues, which do not normally show significant levels of FAS activity. The relative levels of the CAT mRNA driven by the rat FAS promoter in various tissues of the transgenic animals approximated those of the endogenous mouse FAS mRNA. We also examined the hormonal and nutritional regulation of the FAS(2.1)-CAT reporter gene in transgenic mice. CAT activity was increased in both liver and white adipose tissue when fasted animals were refed a high carbohydrate, fat-free diet. These changes in CAT activity and CAT mRNA levels occurred in parallel to the changes in endogenous mouse FAS mRNA levels. On the other hand, fasting/refeeding did not change CAT activity appreciably in other tissues, such as muscle and brown adipose tissue. Administration of dibutyryl cAMP at the start of refeeding prevented an increase in CAT activity in liver. However, the cAMP effect was tissue-specific as cAMP treatment did not bring about change in CAT activity in adipose tissue. Next, to examine the effect of insulin, we made the transgenic mice insulin-deficient by streptozotocin treatment. Insulin treatment of the streptozotocin-diabetic mice increased both the CAT activity and CAT mRNA levels driven by the rat FAS promoter in liver and white adipose tissue. These changes in CAT expression by insulin paralleled those in endogenous FAS mRNA levels. Administration of glucocorticoids increased CAT activity in all tissues examined: liver, white and brown adipose tissues, lung, heart, and spleen. Overall, the first 2.1 kilobases of the 5-flanking region of the rat FAS gene appear to contain sequence elements necessary to confer tissue-specific and hormonally regulated expression characteristic of the endogenous FAS gene. Fatty acid synthase (FAS)1 plays a central role in de novo lipogenesis in mammals and birds by catalyzing all the reactions in conversion of acetyl-CoA and malonyl-CoA to palmitate. FAS activity is not known to be regulated by allosteric effectors or covalent modification. However, FAS concentration in liver and adipose tissue is highly sensitive to nutritional, hormonal, and developmental states (1-3). When rats are fasted for 1-2 days, the rate of synthesis of FAS declines, while refeeding a high carbohydrate diet increases synthesis of FAS (4). We have previously reported that FAS mRNA was not detectable in livers of fasted mice but dramatically increased upon refeeding a high carbohydrate, fat-free diet (5). This induction of FAS mRNA resulted...

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in glycerolipid biosynthesis. We recently cloned a cDNA to a 6.8-kb mRNA, a message that can be induced dramatically by feeding a high-carbohydrate diet [Paulauskis & Sul (1988) J. Biol. Chem. 263, 7049-7054; Shin et al. (1991) J. Biol. Chem. 266, 23834-23839], and identified the open reading frame, p90, as mitochondrial GPAT [Yet et al. (1993) Biochemistry 32, 9486-9491]. To initiate characterization of mitochondrial GPAT, we purified and reconstituted the GPAT activity using phospholipids after expressing functional enzyme in Sf9 insect cells. Infection with recombinant virus containing p90 sequence resulted in high levels of GPAT expression in mitochondria, compared to noninfected cells or cells infected with the reverse orientation insertion baculovirus. There was a dramatic increase in N-ethylmaleimide-resistant mitochondrial GPAT activity. The GPAT protein was not detectable by Western blot in noninfected Sf9 cells or in cells infected with the GPAT sequence in the reverse orientation. However, in cells infected with GPAT in the correct orientation, there was a dramatic increase in the GPAT protein that was readily detectable by Coomassie staining both in total extracts and in the mitochondrial fraction. To ease the purification, we next expressed GPAT as a polyhistidine fusion protein in insect cells. The polyhistidine tag did not interfere with targeting to mitochondria or with the catalytic activity of GPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3-3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.

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