The Saccharomyces cerevisiae protein ELO2p is involved in the elongation of saturated and monounsaturated fatty acids. Among several sequences with limited identity with the S. cerevisiae ELO2 gene, a consensus cDNA sequence was identified from the LifeSeq(R) database of Incyte Pharmaceuticals, Inc. Human liver cDNA was amplified by PCR using oligonucleotides complementary to the 5' and 3' ends of the putative human cDNA sequence. The resulting full-length sequence, termed HELO1, consisted of 897 bp, which encoded 299 amino acids. However, in contrast with the ELO2 gene, expression of this open reading frame in S. cerevisiae demonstrated that the encoded protein was involved in the elongation of long-chain polyunsaturated fatty acids, as determined by the conversion of gamma-linolenic acid (C(18:3, n-6)) into dihomo-gamma-linolenic acid (C(20:3, n-6)), arachidonic acid (C(20:4, n-6)) into adrenic acid (C(22:4, n-6)), stearidonic acid (C(18:4, n-3)) into eicosatetraenoic acid (C(20:4, n-3)), eicosapentaenoic acid (C(20:5, n-3)) into omega3-docosapentaenoic acid (C(22:5, n-3)) and alpha-linolenic acid (C(18:3, n-3)) into omega3-eicosatrienoic acid (C(20:3, n-3)). The predicted amino acid sequence of the open reading frame had only 29% identity with the yeast ELO2 sequence, contained a single histidine-rich domain and had six transmembrane-spanning regions, as suggested by hydropathy analysis. The tissue expression profile revealed that the HELO1 gene is highly expressed in the adrenal gland and testis. Furthermore, the HELO1 gene is located on chromosome 6, best known for encoding the major histocompatibility complex, which is essential to the human immune response.

The enzymes that are involved in the elongation of fatty acids differ in terms of the substrates on which they act. To date, the enzymes specifically involved in the biosynthesis of polyunsaturated fatty acids have not yet been identified. In an attempt to identify a gene(s) encoding an enzyme(s) specific for the elongation of ␥-linolenic acid (GLA) (18:3n-6), a cDNA expression library was made from the fungus Mortierella alpina. The cDNA library constructed in a yeast expression vector was screened by measuring the expressed elongase activity [conversion of GLA to dihomo-GLA (20:3n-6)] from an individual yeast clone. In this report, we demonstrate the isolation of a cDNA (GLELO) whose encoded protein (GLELOp) was involved in the conversion of GLA to dihomo-GLA in an efficient manner (60% conversion). This cDNA contains a 957-nucleotide ORF that encodes a protein of 318 amino acids. Substrate specificity analysis revealed that this fungal enzyme acted also on stearidonic acid (18:4n-3). This report identifies and characterizes an elongase subunit that acts specifically on the two ⌬6-desaturation products, 18:3n-6 and 18:4n-3. When this GLELO cDNA was coexpressed with M. alpina ⌬5-desaturase cDNA in yeast, it resulted in the conversion of GLA to arachidonic acid (20:4n-6) as well as the conversion of stearidonic acid to eicosopentaenoic acid (20:5n-3). Thus, this GLELO gene may play an critical role in the bio-production of both n-6 and n-3 polyunsaturated fatty acids.

Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle ofthe virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-PheAsp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication.The nucleocapsid protein N of vesicular stomatitis virus (VSV) tightly wraps the RNA genome and maintains the structural integrity and the template function of the negativestrand genome RNA (1). Within the virion this N-RNA template is associated with the RNA polymerase L and the transcription factor, phosphoprotein P, to form the transcribing ribonucleoprotein (RNP) complex. During transcription, the RNA polymerase complex (L and P) interacts with the N protein of the N-RNA template to transiently displace N and gain access to the genome RNA. During replication of the genome RNA, a soluble form of the N protein is required (2). This form of the N protein has been proposed to interact with nascently transcribed RNA chains from the RNP complex and to switch the mode of the RNA polymerase from transcription to replication (2). This results in the formation of an N-RNA complex containing full-length sense or antisense genome RNA. The precise mechanism by which the N protein recognizes the cognate sequence within the nascent RNA chain and continues to encapsidate during transcription remains unclear.Development of in vitro replication systems and studies on the structure and function of the N protein have provided some insight into the replication step of the virus' life-cycle (1). Although the N protein alone can initiate replication in vitro, albeit inefficiently, the presence of the P protein was found to greatly stimulate the reaction (3-5). Moreover, itThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact...

An RNA-dependent RNA polymerase is packaged within the virions of purified vesicular stomatitis virus, a nonsegmented negative-strand RNA virus, which carries out transcription of the genome RNA into mRNAs both in vitro and in vivo. The RNA polymerase is composed of two virally encoded polypeptides: a large protein L (240 kDa) and a phosphoprotein P (29 kDa). Recently, we obtained biologically active L protein from insect cells following infection by a recombinant baculovirus expressing L gene. During purification of the L protein from Sf21 cells, we obtained in addition to an active L fraction an inactive fraction that required uninfected insect cell extract to restore its activity. The cellular factors have now been purified, characterized, and shown to be ␤ and ␥ subunits of the protein synthesis elongation factor EF-1. We also demonstrate that the ␣ subunit of EF-1 remains tightly bound to the L protein in the inactive fraction and ␤␥ subunits associate with the L(␣) complex. Further purification of L(␣) from the inactive fraction revealed that the complex is partially active and is significantly stimulated by the addition of ␤␥ subunits purified from Sf21 cells. A putative inhibitor(s) appears to co-elute in the inactive fraction that blocked the L(␣) activity. The purified virions also package all three subunits of EF-1. These findings have a striking similarity with Q␤ RNA phage, which also associates with the bacterial homologue of EF-1 for its replicase function, implicating a possible evolutionary relationship between these host proteins and the RNA-dependent RNA polymerase of RNA viruses.Vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand RNA viruses, has long been a paradigm for studying gene expression of this class of RNA viruses that infect vertebrates, invertebrates, and plants (1). Some of the most common human pathogens that belong to this category are rabies, measles, mumps, and human parainfluenza. A hallmark of all negative strand RNA viruses is the obligate packaging of an RNA-dependent RNA polymerase within the mature virions (2) that transcribes the genome RNA into mRNAs both in vitro and in vivo (3). For VSV, the virion-associated RNA polymerase is generally thought to consist of two virally encoded protein subunits, L (240 kDa) and P (29 kDa), which remain tightly complexed within the virion (3). Studies on the structure and function of VSV RNA polymerase have been greatly aided by the ability to isolate the polymerase subunits from the virions in a relatively pure form (4, 5). Active reconstitution of transcription is achieved by mixing the genome RNA enwrapped with the nucleocapsid protein (N) (referred to as N-RNA template) and purified L and P proteins (4, 5). From a large body of evidence, it appears that the L protein possesses the catalytic activity for RNA synthesis and the P protein is a transcription factor essential for L function (3); no cellular protein(s) has so far been shown to be required for the RNA polymerase activity. Only recently, ...