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EcoRPI restriction endonuclease fragments from a A proviral DNA hybrid containing the entire presumptive avian myeloblastosis virus (AMV) provirus, and from a A proviral hybrid containing a partial myeloblastosis-associated virus type 1 (MAV-1-like provirus were compared by heteroduplex analysis. The cloned presumptive AMV provirus was also analyzed by electron microscopy, using R-loop formation with purified 35S RNA isolated from virions of the standard AMV complex. The results indicate that the putative AMV genome contains a segment absent in its MAV-1-like helper virus. This segment represents a substitution in the region of the genome that in MAV-1 virus is occupied by the envelope gene and is approximately 9004: 160 nucleotide pairs in length. Hybridization of specific probes from the presumptive AMV genome to Southern blots of EcoRI-digested cellular DNA has revealed that these substituted sequences are homologous to chicken and duck DNA that is not related to chicken endogenous proviral sequences.

Clones for rat hepatic lipase were isolated by probing a rat liver cDNA library in Agtll with an oligonucleotide synthesized on the basis of a partial peptide sequence. The cloned messenger codes for a protein of 472 amino acids plus a hydrophobic leader sequence of 22 amino acids. The unglycosylated protein has a predicted molecular weight of 53,222 and contains two potential sites for N-glycosylation. The protein bears striking regions of homology with other known lipases and contains peptide sequences that have been implicated in lipid binding. The homologous mRNA is present in liver tissue but no detectable mRNA is observed in the adrenal gland, despite the reported presence of hepatic lipase in both the liver and the adrenal gland. No mRNA was seen in any of a variety of other tissues.Hepatic lipase, localized primarily on the sinusoidal surfaces of the liver, functions in the metabolism of circulating lipoproteins (1). Hepatic lipase activity has also been detected in several extrahepatic tissues, including adrenal gland and ovary (2-4). The presumed function of the enzyme is the hydrolysis of triglycerides in intermediate density lipoproteins (IDL) and of phospholipids in high density lipoproteins (HDL2) (5). The capacity ofpurified hepatic lipase to catalyze hydrolysis of phospholipids and mono-, di-, and triglycerides (6, 7) is consistent with the putative function. The action of hepatic lipase on HDL2 is presumed to result in the production of HDL3. Also, hepatic lipase may participate in the clearance of circulating very low-density lipoproteins (8) and chylomicron remnants (9).Hepatic lipase from rat liver has been purified to homogeneity (7, 10). The enzyme has an apparent molecular weight of53,000 (7,11) cDNA Screening and Analysis. Partial amino acid sequences were determined from the digestion of purified hepatic lipase with subtilisin (11). A single 42-base oligonucleotide probe was constructed on the basis of a 14 amino acid sequence ( Fig. 1) and codon usage tables for mammalian genes (13). In addition to codon usage assumption, G-T base pairing was allowed in designing the probe. A total of 9 x 105 plaques were screened with the end-labeled 42-base oligonucleotide probe. Plaque replicas on nitrocellulose filters were made by the method of Benton and Davis (14). The filters were baked at 80'C for 2 hr and incubated for 3 hr at 370C in 30% (vol/vol) formamide/900 mM NaCl/90 mM trisodium citrate/5 x concentrated Denhardt's solution/50 mM sodium phosphate, pH 6.5, containing salmon sperm DNA at 100 pkg/ml, in a total volume of 200 ml. The 32P-labeled probe (2 x 108 cpm/Ag) was hybridized to the filters for 17 hr at 370C in 200 ml of the same solution. The filters were washed twice at room temperature in 500 ml of 150 mM NaCl/15 mM trisodium citrate/0.1% NaDodSO4 for 30 min per wash, and then six times at 370C in 300 ml of the same solution for 30 min per wash. The filters were exposed to Du Pont Cronex 4 film with Du Pont Hi-Plus intensifying screens at -70'C for 20 hr. DNA from clones t...

The enzyme hepatic lipase may play several roles in lipoprotein metabolism. Recent investigation has suggested a role for the enzyme in lipoprotein and/or lipoprotein lipid uptake. To study this, a simple isolated system that mimics the in vivo system would be desirable. The enzyme is secreted by the hepatic parenchymal cell but exists, and presumably exerts its effects, while bound to capillary endothelial cells in the liver, adrenal gland, and the ovary. We constructed a cDNA that encodes the expression of a chimeric protein composed of rat hepatic lipase and the signal sequence for the addition of the glycophosphatidylinositol (GPI) anchor from human decay-accelerating factor. When transfected into Chinese hamster ovary (CHO) cells this gave rise to a cell population that had immunoreactive hepatic lipase on the cell surface. Cloning of the transfected cells produced several cell lines that expressed the chimeric protein bound to the cell surface by a GPI anchor. This was documented by demonstrating incorporation of [3H]ethanolamine into anti-hepatic lipase immunoprecipitable material; in addition, hepatic lipase was released from the cells by phosphatidylinositol-specific phospholipase C but not by heparin. Phosphatidylinositol-phospholipase C treatment of cells expressing the anchored lipase released material that comigrated with hepatic lipase on SDS-polyacrylamide gel electrophoresis and was immunoreactive with antibody to the cross-reacting determinant of GPI anchors. Cell lysates containing the anchored protein contained salt-resistant lipase activity, a known feature of the secreted hepatic lipase; thus it appears that these cells have a surface-anchored hepatic lipase molecule. Although it was not possible to demonstrate lipolysis by the enzyme while it was on the cell surface for technical reasons, the protein produced by these cells was active when studied in cell membranes. The ability of the cells to take up lipoproteins was studied. The cells demonstrated an increased affinity for low density lipoprotein (LDL) receptor mediated uptake of LDL. They did not, however, demonstrate any enhanced binding or removal of chylomicron remnants. With respect to LDL and remnants, the cells expressing anchored lipase behaved similarly to CHO cell that expressed secreted hepatic lipase. The cells expressing anchored hepatic lipase had a marked increase in the uptake of high density lipoprotein and high density lipoprotein cholesteryl ester when compared to that seen with CHO cells secreting hepatic lipase. This increase occurred primarily via the selective pathway, and was not reduced by addition of anti-LDL receptor or anti-hepatic lipase antibodies or the receptor-associated protein. Together the results suggest that hepatic lipase, when bound to the cell surface by a GPI anchor, plays a role in enhancing lipoprotein uptake. For LDL this may involve the provision of a second foot for particle binding, thus enhancing affinity for the LDL receptor. For chylomicron remnants an additional molecule or molecules are ...

IntroductionHormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triglycerides within adipocytes and is thought to be the rate limiting enzyme in lipolysis; however, direct evidence to prove this concept has been lacking. The present study was designed to establish the function of HSL in adipocytes. A 2360-bp fragment containing the entire HSL coding region was cloned into the vector pCEP4 and was used to transfect the 3T3-F442A adipogenic cell line. Free fatty acids are a major energy source for most tissues. Circulating FFA in plasma are derived from the breakdown of stored triacylglycerols in adipose tissue (1, 2). It has been suggested that an increased metabolic activity (i.e., a greater release of FFA) of internal fat depots is responsible for the observations linking central obesity with an increased prevalence of hyperlipidemia, atherosclerosis, diabetes mellitus, insulin resistance, and hypertension (3, 4, 5). The major enzyme responsible for the mobilization of FFA from adipose tissue is thought to be hormone-sensitive lipase (HSL),' whose name was coined to reflect the ability of hormones such as catecholamines, ACTH, and glucagon to stimulate the activity of this intracellular neutral lipase (6). HSL catalyzes the first, rate-limiting step in lipolysis by cleaving the first ester-bond of triacylglycerols (7,8). In adipose tissue, HSL also catalyzes the second step of the lipolytic reaction by hydrolyzing diacylglycerol to monoacylglycerol (7,8). Although HSL is capable of hydrolyzing monoacylglycerols to FFA, the final step of the lipolytic process is carried out by another enzyme, monoacylglycerol lipase (9, 10). This is due to the specificity of HSL for 1,(3)-monoacylglycerols, while the main product of the hydrolysis of triacylglycerols is 2-monoacylglycerol (11). Indeed, the findings that monoacylglycerol accumulates in vitro in the presence of HSL when monoacylglycerol lipase is absent- (10)

We have identified and isolated a presumptive leukemogenic provirus from myeloblasts of a chicken in which leukemia had been induced by avian myeloblastosis virus (AMV). Leukemic myeloblasts isolated from peripheral blood or from converted yolk sac cultures of various strains of chickens, regardless of the endogenous proviral content or AMV pseudotype used for infection, contain an EcoRI 2.2-megadalton (MDal) and a HindIII 2.6-MDal proviral fragment. A proviral genome flanked by chicken DNA sequences on either side and containing both the EcoRI 2.2-MDal and the Hindl 2.6-MDal fragments was inserted by molecular recombination into X phage Charon 4A and then cloned. This presumptive AMV proviral genome has a mass of approximately 4.9 MDal and contains terminal redundancies with respect to 3' viral RNA sequences.The standard avian myeloblastosis virus (AMV-S) complex causes acute myeloblastic leukemia, osteopetrosis, visceral lymphoid leukosis, and nephroblastomas in chickens (1). Two cloned avian myeloblastosis-associated viruses, MAV-1 and MAV-2, subgroups A and B, respectively, cause all the neoplasias noted for AMV-S except acute myeloblastic leukemia (2). Thus, an as-yet-unidentified virus in the AMV-S complex is responsible for myeloblastosis. This leukemogenic component (AMV) is thought to be defective because it can induce formation of leukemic myeloblasts in which there is no detectable virus production but from which AMV can be rescued after superinfection with a suitable helper (3). Recently, preparations of AMV-S unintegrated linear proviruses have been shown to contain a viral DNA genome of approximately 4.9 megadaltons (MDal), which is slightly smaller than that of MAV-1 or MAV-2 (5.3 MDal) and could be the genome of the AMV leukemogenic component (4).Three approaches have been taken to further identify the AMV genome: (i) DNA extracted from leukemic myeloblasts isolated from the peripheral blood of leukemic chickens was analyzed for viral DNA sequences after restriction endonuclease digestion; (ii) DNA from cloned myeloblasts converted in vitro by AMV infection of yolk sac cell cultures (5) was analyzed in the same manner as the DNA from the myeloblasts isolated from leukemic chickens; and (iii) an EcoRI partial digest of DNA from leukemic myeloblasts was inserted into X phage Charon 4A by artificial DNA recombination and analyzed for proviral sequences.A presumptive leukemogenic provirus has been identified and isolated from leukemic myeloblasts. It has a molecular mass of approximately 4.9 MDal, the same as that of an unintegrated viral DNA present in preparations of AMV-S linear DNA but absent in linear viral DNA preparations of MAV-1 or MAV-2 (4). MATERIALS AND METHODSChicken Strains and Viruses. The strains and sources of our fertile chicken eggs were: C/E Spafas negative for group specific antigen, chicken helper factor, and virus production (gs-chf-V-) from Spafas (Roanoke, IL), C/O H&N gs-chf-Vfrom H&N Farms (Redmont, WA), C/ABE line 7-2 V+ from the Regional Poultry Research Laborato...

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