African trypanosomes are ancient eukaryotes that cause lethal disease in humans and cattle. Available drugs are inadequate and the need for new therapeutic targets is great. Trypanosoma brucei and related pathogens differ strikingly from higher eukaryotes in many aspects of nucleic acid structure and metabolism. We find yet another example of this in their unusual DNA topoisomerase IB. Type IB topoisomerases relieve the supercoils that accumulate during DNA and RNA synthesis, and are of considerable importance as the target for antitumor camptothecins. Dozens of type IB topoisomerases sequenced from eukaryotes, bacteria, and pox viruses are all encoded by a single gene that predictably contains a highly conserved DNA binding domain and C-terminal catalytic domain, linked by a nonconserved hydrophilic region. We find that topoisomerase IB in T. brucei is encoded by two genes: one for the DNA-binding domain and a second for the C-terminal catalytic domain. In keeping with this, highly purified fractions of native T. brucei topoisomerase IB catalytic activity contain two proteins, of 90 and 36 kDa. The native enzyme is conventional in its Mg 2؉ -independence, ability to relax positive and negative supercoils, and inhibition by camptothecin. Camptothecin promotes the formation of a covalent complex between 32 P-labeled substrate DNA and the small subunit. This unusual structural organization may provide a missing link in the evolution of type IB enzymes, which are thought to have arisen over time from the fusion of two independent domains. It also provides another basis for the design of selectively toxic drug candidates.T he African trypanosome, Trypanosoma brucei, is a flagellated protozoan that causes sleeping sickness in humans. This tsetse fly-transmitted meningoencephalitis is of increasing incidence and is fatal if not treated (1). Closely related organisms cause Chagas' disease and leishmaniasis. Available therapies for sleeping sickness are widely acknowledged to be inadequate: they require multiple parenteral doses for cure, are expensive, toxic, and are losing efficacy against drug-resistant parasites. Trypanosomes and leishmania are ancient eukaryotes whose distinctive features include structurally and metabolically unusual DNAs. In African trypanosomes, the nuclear genome is comprised of 11 chromosome pairs, several additional chromosomes of unknown ploidy, and Ϸ100 minichromosomes, each containing a single gene that encodes a variable surface protein (2). Surface protein genes can be activated by recombinationbased transfer from minichromosomes to transcriptionally active sites in larger chromosomes. Large polycistronic transcripts are trans-spliced to form mRNAs with a 5Ј-leader sequence and 3Ј poly(A) tail. Even more unusual is their mitochondrial DNA, termed kinetoplast or kDNA, which is in the form of a single gigantic network of interlocked relaxed DNA circles (3, 4). Mature mitochondrial messages are created by an editing process that involves systematic insertion or removal of U residues.DNA top...

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Based on transfection into cells in culture or natural transduction into retroviruses, proto-ras genes seem to derive transforming function either from heterologous promoters or from point mutations. Here we ask how such different events could achieve the same results. To identify homologous regulatory elements, about 3 kilobases of rat DNA upstream of the first untranslated proto-Ha-ras exon was sequenced. Surprisingly, the sequence shares at -1858 a homology of 148 nucleotides with Harvey (Ha) sarcoma virus, 5' of viral ras, signaling possibly a second untranslated proto-Ha-ras exon. In addition the sequence contains a perfect repeat of 25 CA dinucleotides at -2655. A retroviral promoter, even from upstream of the poly(CA), conferred transforming function on proto-Ha-ras and increased transcription >100-fold compared with that of unrearranged proto-ms. Point mutations were not necessary for transforming function of rat and human protoHa-ras genes with retroviral promoters but did enhance it >10-fold. A unifying hypothesis proposes that proto-ras genes depend on high expression from heterologous promoters or enhancers for transforming function, which is modulated by ras point mutations. The hypothesis makes two testable predictions.(a) Unrearranged proto-ras genes with point mutations, which occur in some cancers, have no transforming function. Indeed, tumors with mutated proto-ras genes, even those that also lack hypothetical tumor-suppressor genes, are indistinguishable from counterparts with normal proto-ras genes. (is) Proto-ras genes in transfected cells derive transforming function from heterologous promoters or enhancers acquired via illegitimate recombination from vector DNAs and particularly from viral helper genes that must be cotransfected for transformation of primary cells. Indeed, expression of exogenous proto-ras genes in cells transformed by transfection is as high as for viral ras genes and is much higher than in the cells of origin.Harvey (Ha), BALB, Rasheed, and two other murine sarcoma viruses carry autonomous Ha-ras genes that cause tumors in animals and transform cells in culture (1-5). Kirsten sarcoma virus carries a structurally distinct but functionally very similar Ki-ras gene (1, 5). All viral ras (v-ras) genes differ from normal, cellular proto-ras genes in virus-specific promoters and virus-specific point mutations of their common ras coding region (2-9). Based on site-directed mutagenesis we (8, 9) and others (10) have shown that retroviral promoters are essential, and that point mutations are not necessary, for transforming function of v-ras genes.Based on transfection into cells in culture, proto-ras genes appear to derive transforming function either from specific point mutations (11-13) or from heterologous promoters and enhancers (8)(9)(10)(14)(15)(16). For example, upon transfection, point-mutated proto-ras genes from some tumors transform the mouse NIH 3T3 line directly (11, 12) and transform primary rodent cells in conjunction with retroviral or DNAviral helper gene...

The pattern of human retinoblastoma (RB1) gene protein expression was directly examined in normal and malignant human colorectal tissues and in seven colorectal carcinoma cell lines by immunohistochemistry using the mouse monoclonal antibody (RB-MAb-1) directed against the retinoblastoma protein (RB). This is the first demonstration of RB immunostaining in adult human colonic epithelium and colorectal carcinomas. Specificity using RB-MAb-1 was confirmed by western blot analysis, which showed bands of 110-116 kDa corresponding to the sizes of unphosphorylated and phosphorylated RB. RB staining of normal adult colonic epithelium was confined to the nucleus and was most intense in the transitional zone of the crypt, whereas lumenal cells (fully differentiated) were RB negative. Primary colorectal carcinomas and all the colon cancer cell lines stained positively for nuclear RB, but the expression was heterogeneous with varying fractions of RB negative cells present. Because we and others have previously shown that loss or inactivation of the RB1 gene is infrequent in colorectal carcinomas, reduced RB expression in such cells is probably due to a cellular regulatory mechanism. For example, RB negative cells may be those in early-G1 phase (known to have reduced RB levels) or growth-arrested cells that have differentiated. The ability to directly detect RB in primary colorectal carcinomas will permit assessment of whether heterogeneous expression of the RB1 gene product has prognostic significance for survival of patients with this cancer.