Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.

Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide by molecular oxygen; long-chain aldehyde is required for light emission. At 200 the bioluminescence has a lifetime of tens of seconds, while excess reduced flavin is removed by way of nonenzymatic autoxidation in less than a second. This observation indicates the existence of a long-lived enzyme intermediate, which has been postulated to be a peroxide of the enzyme-bound reduced flavin. This intermediate was isolated and studied at low temperature (-20°) emission of light, an increase in both absorption (450 nm) and fluorescence emission (530 nm) occurred.The most important requirement for a detailed study of the enzyme intermediate is that it be separated from the free flavin. In recent years, techniques for studying enzymes and their reactions at low temperatures have been developed in our laboratory (3). Since the lifetime of intermediate II at low temperatures (-20°to -50°) is measured in hours or days (4), separation by column chromatography at -20°was attempted and achieved. Intermediate II was found to have an absorption maximum at 370 nm and a fluorescence emission peaking at 485 nm, consistent with the hypothesis that it involves a flavin-peroxide (5). MATERIALS AND METHODSBacterial luciferase, isolated from Achromobacterfischeri, strain MAY (6), was purified-(7) and stored at -20°before use.Since it was prepared by a short procedure, there remained some impurities, especially some absorbance in the visible range. These were not large enough, however, to interfere with the observations. Its specific activity with FMNH2, determined at the time these experiments were done, was 4.6 X 10'3 quanta sec-' mg-1, with dodecanal at 220. Electrophoresis on acrylamide gels indicated a purity of about 80%. Bioluminescence was measured with a photometer (8) calibrated with the standard of Hastings and Weber (9).As already described for other enzymes (3), a medium appropriate for studies at low temperatures must be an innocuous organic solvent mixed with an aqueous system to depress the freezing point, and adjusted to suitable ionic environment and proton activity. For luciferase, a suitable medium in which the enzyme is active and not denatured at low temperatures is 50%/, ethylene glycol-lQ mM phosphate buffer (pH 7) (10). Under these conditions the actual "proton activity" (pH*) of the medium is 7.6 at +200 and 7.8 at -20° ( 11)