Extensive amplification and transposition of a novel repetitive element, xstir, together with its terminal inverted repeat in the evolution of Xenopus.

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis.

Role of type III iodothyronine 5-deiodinase gene expression in temporal regulation of Xenopus metamorphosis.

To elucidate the role of type III iodothyronine 5-deiodinase (5-D) in the temporal regulation of amphibian metamorphosis, the regulation of gene expression of 5-D and thyroid hormone receptor beta (TRbeta) in organs of Xenopus laevis was investigated. High levels of TRbeta mRNA in the respective organs were observed at the times of their major morphological changes. Expression of the 5-D gene was highly regulated among the organs during metamorphosis, including up-regulation in the tail and down-regulation in the liver. The tail and liver expressed 5-D gene before their metamorphic changes. These precocious expressions correlated with the lower responsiveness to exogenously added triiodo-L-thyronine (T3) for inducing a high level of TRbeta mRNA expression. However, the same organs responded to lower doses of T3 to regulate 5-D gene expression as seen in spontaneous metamorphosis. The induction of 5-D gene expression was considerably delayed in the intestine, even at an excess dose of T3. Thus, the two genes in a given organ appeared to respond to T3 either with different dose dependencies or with different timetables. The results obtained are also discussed in respect to recent findings in Rana catesbeiana.

Coexpression and promoter function in two muscle actin gene complexes of different structural organization in the ascidian Halocynthia roretzi.

During embryogenesis of the ascidian Halocynthia roretzi, 42 unicellular striated muscle cells are formed in the tail of the tadpole larva. Isolation of cDNA clones demonstrated that multiple genes for larval muscle actin are expressed in this process. Among them, at least five muscle actin genes (HrMA2, HrMA4a, HrMA4b, HrMA5, and HrMA6) form a cluster (HrMA2/4 cluster) within about 30 kb of the genome. The 5' flanking sequences of the five actin genes resemble each other. When constructs in which 184 bp of the 5' flanking region of each of these genes fused with lacZ were introduced into fertilized eggs, the reporter gene was expressed in muscle cells of the tailbud embryo, suggesting that the 5' flanking region of each cluster gene has promoter activity. In addition, a pair of muscle actin genes, HrMA1a and HrMA1b (HrMA1 pair), was isolated from a genomic region different from that of the HrMA2/4 cluster. The HrMA1a and HrMA1b are linked in a head-to-head arrangement on opposite strands and share a 340-bp 5' flanking sequence containing two symmetrically located TATA boxes. HrMA1a showed basically the same expression pattern as that of HrMA4a. When constructs in which the shared upstream region of HrMA1 pair fused with lacZ in either direction were microinjected into eggs, the reporter gene was expressed in muscle cells of the larval tail, suggesting a bidirectional promoter that regulates muscle-specific transcription of the HrMA1 pair. The tandem cluster of HrMA2/4 genes and the bidirectional promoter of the HrMA1 pair could expedite utilization of muscle-specific trans-acting factors. The organization of genes in the genome may play an important role in the synthesis of a large amount of actins during the process of rapid differentiation.

Short upstream sequences associated with the muscle-specific expression of an actin gene in ascidian embryos.

The HrMA4 alpha gene for an embryonic muscle actin of the ascidian Halocynthia roretzi is activated at the gastrula stage in differentiating muscle cells exclusively. The 5' upstream region close to the transcription start site of HrMA4 alpha contains several consensus sequences, which include a TATA box at -30, and E-box at -71, a CArG box at -116, and a cluster of three E-boxes between -150 and -190. When deletion constructs of this region, fused with the bacterial gene for beta-galactosidase (lac-Z), were microinjected into fertilized eggs, the reporter gene was expressed in muscle cells of tailbud embryos. Analyses of the deletion constructs suggested that the 103-bp upstream region is sufficient for the appropriate expression of the gene. However, beta-gal activity was very rarely detectable in the case of 82-bp upstream region and no activity was detected in the case of 72-bp upstream region. Mutations in the proximal E-box sequence did not disturb the muscle-specific expression of the reporter gene. These results suggest that rather short sequences between nucleotides -103 and -72 from the transcription start site are associated with the specific expression of HrMA4 alpha.

[Dose intensity in M-VAC chemotherapy with rG-CSF for metastatic transitional urothelial cancer].

The effect of M-VAC (methotrexate, vinblastine, adriamycin and cisplatin) chemotherapy supported recombinant human granulocyte stimulating factor (rG-CSF) was studied in 18 patients with metastatic urothelal cancer. The mean age of the patients was 66 years. Of the patients 6 had lung metastasis and 12 had distant lymph node metastasis. In this study recombinant human granulocyte stimulating factor was administered therapeutically at 100 to 250 micrograms subcutaneously after the white blood cell count was less than 3,000/mm. Relative dose-intensity (RDI) was 0.88 +/- 0.11 and 0.72 +/- 0.19 for 11 patients with rG-CSF and 7 without rG-CSF, respectively. The relative dose regardless of the interval of the cycle of M-VAC chemotherapy (%Dose) was 0.95 +/- 0.06 and 0.86 +/- 0.09 for the patients with rG-CSF and those without rG-CSF, respectively. Of seven patients without rG-CSF 3 patients responded (response rate 42%, mean survival period 7.4 months). Of 11 patients treated with rG-CSF 3 patients responded (response rate 72.7%, mean survival period 20.0 months). Although rG-CSF increased the RDI of M-VAC chemotherapy, the correlation of response with RDI was not clearly demonstrated. The therapeutical administrations of rG-CSF fail to improve the mean white blood cell nadir and to prevent the decrease of the platelet and reticulocyte count.