A systematic search for upstream controlling elements necessary for efficient expression of the yeast fatty acid synthase genes FAS1 and FAS2 revealed identical activation sites, UASFAS, in front of both FAS genes. The individual element confers, in a heterologous yeast test system, an approximately 40‐fold stimulation of basal gene expression. The UASFAS motifs identified have the consensus sequence TYTTCACATGY and function in either orientation. The same sequence motif is found in the upstream regions of all so far characterized yeast genes encoding enzymes of phospholipid biosynthesis. In gel retardation assays, a protein factor, Fbf1 (FAS binding factor), was identified which interacted with UASFAS. The UASFAS motif proved to be an inositol/choline responsive element (ICRE) conferring strict repression by exogenous inositol and choline on a heterologous reporter gene. Its core sequence perfectly matches the CANNTG motif typical of basic helix‐loop‐helix DNA‐binding proteins. In contrast to the individual UASFAS element, the intact yeast FAS promoters are not significantly influenced by inositol and choline, and thus allow nearly constitutive fatty acid synthase production. Available evidence suggests that additional cis‐ and trans‐acting elements, other than UASFAS and Fbf1, are involved in this constitutive FAS gene expression.

The Brevibacterium ammoniagenes fatty acid synthetase (FAS) gene was isolated from a series of overlapping clones by both immunological and plaque hybridization screening of two independent gene libraries. From the isolated DNA a contiguous segment of 10,549 bp was sequenced in both directions. The sequenced DNA contained a very long (9312 nucleotides) open reading frame coding for a protein of 3104 amino acids and with a molecular mass of 327,466 daltons. Based on characteristic sequence motifs known from other FAS systems, seven different FAS active centres were identified at distinct locations within the polypeptide chain. Only one component enzyme, the 3-hydroxydecanoyl beta, gamma-dehydratase, has not yet been localized definitively within the gene. Translation is presumed to start from a GUG triplet located 25 nucleotides downstream of the transcriptional initiation site. There is a canonical Shine-Dalgarno sequence just before this start codon. Comparison of the B. ammoniagenes FAS sequence with those of other known fatty acid synthetases revealed a particularly high degree of similarity to the products of the two yeast genes, FAS1 and FAS2 (30% identical and 46% identical plus closely related amino acids). This similarity extends over the entire length of the genes and involves not only the primary sequences of individual component enzymes but also their sequential order within the multifunctional proteins. These data, together with those on the structure of other fatty acid synthetases, are interpreted in terms of a contribution of both primary structure and subunit cooperation to a conserved topology of functional domains common to all type I FAS complexes.

The fatty acid synthase genes FASl and FAS2 of the yeast Saccharomyces cerevisiae are under transcriptional control of pathway-specific regulators of phospholipid biosynthesis. However, sitedirected mutagenesis of the respective cis-acting elements upstream of FASl and FAS2 revealed that additional sequences activating both genes must exist. A deletion analysis of the FASl promoter lacking the previously characterized inositolkholine-responsive-element motif defined a region (nucleotides -760 to -850) responsible for most of the remaining activation potency. Gel-retardation experiments and in-vitro DNase footprint studies proved the binding of the general regulatory factors Raplp, Abflp and Reblp to this FASI upstream region. Mutation of the respective binding sites led to a drop of gene activation to 8% of the wild-type level. Similarly, we also demonstrated the presence of a Reblp-binding site upstream of FAS2 and its importance for gene activation. Thus, in addition to the previously characterized FAS-binding factor 1 interacting with the inositolkholineresponsive-element motif, a second motif common to the promoter regions of both FAS genes could be identified. Transcription of yeast fatty acid synthase genes is therefore subjected to both the pathway-specific control affecting genes of phospholipid biosynthesis and to the activation by general transcription factors allowing a sufficiently high level of constitutive gene expression.

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO_ operator fragment was inserted at three different positions in front of a tRNAGrU(Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a 1-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 ,g/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in f-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.tRNA suppressors are a classic means of regulating the expression of a protein. In the absence of a suppressor tRNA, the mRNA derived from a gene which has been mutated to contain a stop codon will be incompletely translated. When a tRNA suppressor gene is introduced into the cell, an authentic protein is synthesized, provided the suppressor tRNA inserts the same amino acid as the cognate tRNA of the unmutated codon. This is a powerful tool for analysis of the function of any gene but has been relegated primarily to procaryotes because of the difficulty of manipulating tRNA expression in eukaryotic cells. Eukaryotic tRNA genes contain gene-internal polymerase III (polIII) promoters (for reviews, see references 23 and 46). These transcriptional control regions become part of the mature tRNA coding sequence and are indispensable for tRNA function. As a consequence, these regions are difficult to manipulate. Another consequence is that other than in the case of polII genes, polIII gene promoters cannot be replaced by regulated polII promoters, such as the heat shock and metallothionein promoters.In addition to the gene-internal control elements, 5'-flanking regions of tRNA genes frequently exert a modulatory influence on gene expression (1-3, 10, 15, 17, 27, 37, 41, 47, 49). Although the mechanism of tRNA gene modulation by 5'-flanking regions is not understood, we recently demonstrated that stable binding of a protein near a tRNA gene strongly inhibits its expression (39). This inhibition apparently resulted from masking of the initiation site for tRNA gene transcription by the bound protein and from a strong interaction of the protein with the RNA polIII transcription complex. These findings suggested th...

To explore the ability to use genetic fusions of transferrin as a carrier for brain targeting and delivery, a series of fusion proteins containing both human nerve growth factor (NGF) and human transferrin was produced in mammalian cells. A protein in which the hinge region from human IgG3 joined the carboxyl terminus of NGF and the amino terminus of transferrin formed a covalent homodimer, bound human transferrin receptor, and retained full NGF in PC12 cells. In contrast, proteins in which polypeptide dimerization was not induced or in which NGF was fused through its amino terminus had greatly reduced NGF activity. The ability to maintain both biologically active NGF and transferrin as part of a fusion protein may offer a novel way to deliver NGF and other neurotrophic factors to the central nervous system.