We investigated the changes in gene expression accompanying the development and progression of kidney cancer by use of 31,500-element complementary DNA arrays. We measured expression profiles for paired neoplastic and noncancerous renal epithelium samples from 37 individuals. Using an experimental design optimized for factoring out technological and biological noise, and an adapted statistical test, we found 1738 differentially expressed cDNAs with an expected number of six false positives. Functional annotation of these genes provided views of the changes in the activities of specific biological pathways in renal cancer. Cell adhesion, signal transduction, and nucleotide metabolism were among the biological processes with a large proportion of genes overexpressed in renal cell carcinoma. Down-regulated pathways in the kidney tumor cells included small molecule transport, ion homeostasis, and oxygen and radical metabolism. Our expression profiling data uncovered gene expression changes shared with other epithelial tumors, as well as a unique signature for renal cell carcinoma. [Expression data for the differentially expressed cDNAs are available as a Web supplement at http://www.dkfz-heidelberg.de/abt0840/whuber/rcc.]

We have used a reconstituted cell-free transcription system to investigate the molecular basis of mitotic repression of RNA polymerase I (pol I) transcription. We demonstrate that SL1, the TBP-containing promoter-binding factor, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivated by dephosphorylation. Transcriptional inactivation in vitro is accompanied by phosphorylation of two subunits, e.g. TBP and hTAF I 110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAF I 110 or both, SL1 was purified from two HeLa cell lines that express either full-length or the core domain of TBP only. Both TBP-TAF I complexes exhibit similar activity and both are repressed at mitosis, indicating that the variable N-terminal domain which contains multiple target sites for cdc2/cyclin B phosphorylation is dispensable for mitotic repression. Protein-protein interaction studies reveal that mitotic phosphorylation impairs the interaction of SL1 with UBF. The results suggest that phosphorylation of SL1 is used as a molecular switch to prevent pre-initiation complex formation and to shut down rDNA transcription at mitosis.

The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis, are oligosaccharide antibiotics. They consist of 17 monosaccharide units and the unique aglycon N-(m,p-dihydroxycinnamoyl)taurine. To investigate candidate genes responsible for the formation of trans-m,p-dihydroxycinnamic acid (caffeic acid) as part of the saccharomicin aglycon, gene expression experiments were carried out in Streptomyces fradiae XKS. It is shown that the biosynthetic pathway for trans-caffeic acid proceeds from L-tyrosine via trans-p-coumaric acid directly to trans-caffeic acid, since heterologous expression of sam8, encoding a tyrosine ammonia-lyase, led to the production of trans-p-hydroxycinnamic acid (coumaric acid), and coexpression of sam8 and sam5, the latter encoding a 4-coumarate 3-hydroxylase, led to the production of trans-m,p-dihydroxycinnamic acid. This is not in accordance with the general phenylpropanoid pathway in plants, where trans-pcoumaric acid is first activated before the 3-hydroxylation of its ring takes place.Saccharothrix is a genus of gram-positive bacteria belonging to the well-known order Actinomycetales. Most agents used at present for the treatment of bacterial infections were discovered in members of the Actinomycetales. Saccharothrix espanaensis produces the two heptadecaglycoside antibiotics saccharomicins A and B, which represent a new class of antibiotics (15, 18). They exhibit potent antibacterial activity both in vitro and in vivo against multiply-resistant strains of Staphylococcus aureus as well as vancomycin-resistant enterococci (25). The saccharomicins consist of an oligosaccharide portion and the intriguing aglycon N-(m,p-dihydroxycinnamoyl)taurine (Fig. 1), in which caffeic acid is linked to the amino sulfonic acid taurine via an amide bond.Enzymes belonging to the group of ammonia-lyases catalyze the conversion of ␣-amino acids into ␣,␤-unsaturated acids by elimination of ammonia. Ubiquitous in plants and fungi, phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) catalyzes the nonoxidative deamination of the primary amino acid L-phenylalanine to trans-cinnamic acid (trans-cinnamate), which is the first reaction of the so-called general phenylpropanoid pathway in plants (8). Phenylpropanoids include several important natural product classes, for example, flavonoids, lignins, and coumarins. In monocotyledons, PAL utilizes L-tyrosine in addition to L-phenylalanine (resulting in trans-p-coumaric acid), whereas the enzyme from dicotyledons converts only L-phenylalanine sufficiently. Both PAL and tyrosine ammonia-lyase (TAL) activity are very rare in bacteria (17, 30).The next reactions of the three-step general phenylpropanoid pathway are catalyzed by the enzymes trans-cinnamate 4-monooxygenase (also called cinnamate 4-hydroxylase; EC 1.14.13.11), leading to trans-p-coumaric acid (trans-4-coumarate), and 4-coumarate-coenzyme A (CoA) ligase (EC 6.2.1.12), leading to 4-coumaroyl-CoA (8).In this report, we describe the cloning and identification of two genes from S. espanaensis w...

The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.

The entire simocyclinone biosynthetic cluster (sim gene cluster) from the producer Streptomyces antibioticus Tü6040 was identified on six overlapping cosmids (1N1, 5J10, 2L16, 2P6, 4G22, and 1K3). In total, 80.7 kb of DNA from these cosmids was sequenced, and the analysis revealed 49 complete open reading frames (ORFs). These ORFs include genes responsible for the formation and attachment of four different moieties originating from at least three different pools of primary metabolites. Also in the sim gene cluster, four ORFs were detected that resemble putative regulatory and export functions. Based on the putative function of the gene products, a model for simocyclinone D8 biosynthesis was proposed. Biosynthetic mutants were generated by insertional gene inactivation experiments, and culture extracts of these mutants were analyzed by high-performance liquid chromatography. Production of simocyclinone D8 was clearly detectable in the wild-type strain but was not detectable in the mutant strains. This indicated that indeed the sim gene cluster had been cloned.Simocyclinone D8 (Fig. 1) is produced by Streptomyces antibioticus Tü6040. It is active against gram-positive bacteria and also shows distinct cytostatic activities against human tumor cell lines (39,40,46). Simocyclinone D8 consists of four different moieties, an angucyclic polyketide core, a deoxyhexose (D-olivose), a tetraene side chain, and a halogenated aminocoumarin. The aromatic polyketide moiety is characterized by a large number of unusually placed hydroxyl groups and an oxiran bridge at positions C-12a and C-6a. It contains a Cglycosidically linked D-olivose at position C-9. Attached to the 4-OH group of D-olivose is an acetyl group, and attached to the 3-OH group is a tetraene side chain. Both are linked to the deoxysugar by ester bonds. The final amino-coumarin moiety is linked to the tetraene chain by an amide bond, resulting in an unusual polyene-amide structure. Features that distinguish simocyclinone from other angucycline antibiotics are the enormous size of the molecule and the fact that it originates from at least three different pools of primary metabolites. S. antibioticus Tü6040 also produces other simocyclinones, which can be seen as intermediates of simocyclinone D8. These compounds include simocyclinones of the A-series, the B-series, and the C-series, consisting either of the polyketide moiety (series A), the polyketide moiety plus D-olivose (series B), and the polyketide moiety plus D-olivose plus the tetraene side chain (series C) (40). Genetic engineering and combinatorial biosynthesis in bacteria provide an important new tool for drug discovery and drug design (16,19,24). Knowledge of the sequence and function of genes involved in the biosynthesis of natural products is prerequisite for this new approach. In the present study we describe the isolation of the simocyclinone biosynthetic gene cluster. Sequencing of the entire gene cluster revealed the presence of 49 open reading frames (ORFs) probably involved in simocyclinone bios...

Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus Tü 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.

Phenalinolactones are terpene glycosides with antibacterial activity. A striking structural feature is a highly oxidized gamma-butyrolactone of elusive biosynthetic origin. To investigate the genetic basis of the phenalinolactones biosynthesis, we cloned and sequenced the corresponding gene cluster from the producer strain Streptomyces sp. Tü6071. Spanning a 42 kbp region, 35 candidate genes could be assigned to putatively encode biosynthetic, regulatory, and resistance-conferring functions. Targeted gene inactivations were carried out to specifically manipulate the phenalinolactones pathway. The inactivation of a sugar methyltransferase gene and a cytochrome P450 monoxygenase gene led to the production of modified phenalinolactone derivatives. The inactivation of a Fe(II)/alpha-ketoglutarate-dependent dioxygenase gene disrupted the biosynthetic pathway within gamma-butyrolactone formation. The structure elucidation of the accumulating intermediate indicated that pyruvate is the biosynthetic precursor of the gamma butyrolactone moiety.

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