Semisynthetic promoters activated by cyclic AMP receptor protein of Escherichia coli.

Semisynthetic promoters activated by Escherichia coli cyclic AMP receptor protein (CRP) were created by combining a synthetic CRP-binding site (crb) and nucleotide sequences derived from cryptic promoter regions. A 22-bp oligodeoxyribonucleotide corresponding to an idealized crb was randomly placed into DNA regions that precede a promoterless lacZ gene on a plasmid. Several plasmid clones were obtained which allowed the expression of lacZ in crp+ cya+ cells carrying a chromosomal deletion of lac genes. The beta-galactosidase and the quantitative S1-nuclease assays of crp+ and delta crp cells harboring these plasmids indicated that the transcription from newly created promoters is dependent on CRP. Sequence analysis revealed that these promoters are divided into two types based on the location of the crb relative to the transcription start point (tsp). The distance from the center of the crb to the tsp is 70 bp in the first type and 38 bp in the second type. The sequences of all these promoters exhibit poor homology with the consensus promoter sequence.

Molecular evaluation of bone marrow involvement in peripheral T-cell lymphoma with a PCR-mediated RNase protection assay.

Bone marrow (BM) involvement in peripheral T-cell lymphoma was assessed by polymerase chain reaction (PCR)-mediated RNase protection assay. The sensitivity of this assay was approximately 10(-4) to 10(-5). In 16 of 30 patients (53.3%) with peripheral T-cell malignancies, consensus primers for the T-cell receptor (TCR)-gamma gene amplified the rearranged V(N)J region. Using the PCR products of diagnostic lymph nodes of the patients as probes, we analyzed the BM involvement of lymphoma cells in eight patients: four with peripheral T-cell lymphoma, unspecified; two with adult T-cell leukemia/lymphoma; and two with angioimmunoblastic T-cell lymphoma. BM involvement was detected by PCR-mediated RNase protection assay in four patients from BM smear and/or histo-pathological examination of clotted BM. Moreover, in two of four patients in whom BM involvement was not evident from morphological examination, BM involvement was detected by PCR-mediated RNase protection assay. Our results indicate that the PCR-mediated RNase protection assay targeting the TCR-gamma gene is useful in detecting minimal residual disease in about half of all T-cell lymphoma cases. In addition, in some patients with peripheral T-cell lymphoma, morphologically unproven BM involvement was found using the method.

Evaluation of seven alternative assays on the main ingredients in cosmetics as predictors of Draize eye irritation scores.

This study evaluates seven alternative assays carried out on the main ingredients in cosmetics to determine which battery is the best set and what is the best predictor of the maximal Draize rabbit eye irritation scores (MDESs). The assays consisted of the maximal primary Draize rabbit skin irritation scores (MDSSs), a cytotoxicity test on neutral red uptake using Chinese hamster lung cells (NR-EC(50)), a cytotoxicity test on MTT using normal skin fibroblasts (MTT-EC(50)), the hen's egg test-chorioallantoic membrane test using fertile chicken eggs (HET-CAM), a haemolysis test using red blood cells from Wistar rats (HC(50)), a protein denaturation test using haemoglobin from bovine (HDR), and pH. We tested 10% solutions of 24 tested chemicals, that is, 20 surfactants, three solvents and formaldehyde, to select from the assays a best set for prediction (x) and to obtain the best predictor [f(x)] based on the prediction sum of squares criterion. The selected set consisted of NR-EC(50) and HDR, and the resultant best predictor was f(x) = 74.0 - 29.52 log(NR-EC(50)) + 0.87 HDR. This predictor achieved a high score of 89.6% of the contribution ratio. For two of the 24 test chemicals, an inconsistency occurred in the criticality of corneal damage between the observed and predicted Draize eye scores, although this is not considered to affect significantly the overall results.

[Therapy-related leukemia with t(8;21) initially diagnosed as MDS (RAEB in T)].

A 64-year-old woman was diagnosed as having myelodysplastic syndrome (MDS) at 45 months after receiving radiotherapy for advanced carcinoma of the uterine cervix. We chose low dose therapy of SPAC and ACR because of the diagnosis as therapy-related MDS and the existence of radiation colitis. She obtained minor response, but two months later she transformed to AML (M2). The interval between low dose therapies was getting shorter and shorter, so we tried intensive chemotherapy consisting of BHAC, ACR and 6MP. Blast numbers were reduced, but she died of sepsis and intestinal bleeding. The patients of MDS with t(8;21) and the patients of therapy-related AML (tAML) with t(8;21) are very rare. According to the literature, only karyotype is a prognostic factor in AML/MDS with t(8;21). And diagnosis by the criteria of FAB classification is of little value regarding clinical progress. That is to say, if the patient has only t(8;21) or karyotypic abnormalities which are of little value in prognosis, such as the loss of a sex chromosome, it must be treated as de novo AML, but if patient has karyotypic abnormalities such as -5, 5q-, -7, 7q-, and/or multiple (complicated) abnormalities, we must accept that the prognosis is poor and must treat it as ordinary MDS/tAML.

[Infiltration into the aqueous humor (uveitis) in a patient with malignant lymphoma].

An 80-year-old male was admitted to our hospital because of multiple tumors in October 1989. A pathological diagnosis of non-Hodgkin's lymphoma (diffuse, medium-sized cell type) was made with the histological examination of his biopsied tumors. His clinical stage was stage IV A. Chemotherapy (CHOP) brought him to complete remission. In May 1990, relapse of non-Hodgkin's lymphoma was observed in the neck and both eyes. The diagnosis of uveitis due to involvement of lymphoma cells was confirmed by an aspiration biopsy of left aqueous humor. Complete recovery of his visual acuity was possible with additional chemotherapy, and lymphoma cells in his right aqueous humor were expelled completely, while his cervical tumor remained 1/3 of the original size. He eventually died of pneumonia on December 9th, 1990. Intraocular involvement of malignant lymphoma is rare in comparison with extraocular involvement. Especially, the patient who has only turbid aqueous humor as an ophthalmic sign is very rare. In this paper, we reported a case of lymphoma with intraocular involvement and discussed the mechanisms of infiltration of lymphoma cells into the aqueous humor and the therapy.

[Two patients with aplastic anemia successfully treated with G-CSF and erythropoietin].

Two patients with severe aplastic anemia received combination therapy consisting of granulocyte colony-stimulating factor (G-CSF) and erythropoietin (EPO) by subcutaneous injection. During the first 8 weeks, we administered only G-CSF, but subsequently we administered EPO with G-CSF. Administration of G-CSF caused rapid increase in neutrophil counts in each case. In the first case, a 23-year-old woman, the first sign of improvement of anemia appeared in the 12th week but 50 more weeks elapsed before improvement of platelet count. In the second case, a 59-year-old woman, marked increase of reticulocytes appeared in the 32nd week, and the RBC count become normal in the 46th week. Minor improvement of platelet count was obtained in the 40th week. After 53 weeks of treatment, we stopped administration of G-CSF and EPO, and all of peripheral blood cell counts decreased. Therefore G-CSF and EPO were given to her again, and she showed the same response as first administration. It showed that she was dependent upon G-CSF and EPO. Finally she obtained normalization of all blood cell counts. In both cases, no serious side effects were observed. Combination therapy consisting of G-CSF and EPO may be beneficial for patients with aplastic anemia.

Molecular mechanism of negative autoregulation of Escherichia coli crp gene.

Transcription of the Escherichia coli crp gene encoding cAMP receptor protein (CRP) is negatively regulated by CRP-cAMP complex that binds to a specific site located downstream from the transcription start site. The binding of CRP-cAMP to this site activates transcription from a second divergent overlapping promoter. The mechanism of this negative autoregulation of the crp gene has been investigated by in vitro transcription, gel shift, DNase I footprinting, and exonuclease III protection assays. We demonstrated that the crp and divergent promoters are reciprocally and coordinately regulated by CRP-cAMP. The abortive initiation assay revealed that the divergent RNA itself is not required for the inhibition of crp transcription. Detailed binding studies revealed that CRP-cAMP stimulates the binding of RNA polymerase to the divergent promoter and thus blocks the occupation of the crp promoter by RNA polymerase.

A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation.

Transcription of the Escherichia coli crp gene is negatively regulated by CRP-cAMP that binds to a specific site located downstream of the crp promoter. A second binding site for CRP-cAMP (CRP site II) exists upstream of the crp promoter. Using an in vitro transcription assay, we have demonstrated that CRP-cAMP activates transcription of crp in certain conditions. A promoter which carries an altered CRP-binding site II is no longer activated by CRP-cAMP, indicating that CRP site II mediates the activation of crp transcription. The concentrations of cAMP that are required for positive autoregulation are higher than those for negative autoregulation. Evidence for positive and negative autoregulation in vivo is presented by a quantitative S1 nuclease analysis.

Transcriptional terminator is a positive regulatory element in the expression of the Escherichia coli crp gene.

Plasmids were constructed that contain deletions in the stem region of the presumed rho-independent terminator of the cloned crp gene of Escherichia coli. The level of cyclic AMP binding activity and the amount of CRP in cells harboring the deletion plasmids were found to be significantly lower than those in cells harboring the wild-type crp plasmid. Quantitative S1 assays indicated that the steady-state levels of crp mRNA were markedly reduced in cells harboring the deletion plasmids. Evidence was also presented to show that the crp mRNAs produced from deletion plasmids are less stable than that from the intact crp gene. In vitro transcription assays revealed that the putative crp terminator is indeed a rho-independent terminator. Using the galK expression system and Northern blot analysis we showed that the crp terminator is functional in vivo. Moreover it was shown that the deletion mutations in the stem region of the crp terminator cause a significant readthrough. We conclude that the 3'-flanking sequence of the crp gene acts to stabilize its own mRNA as well as to terminate transcription.

A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli.

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and beta-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on beta-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, beta-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.

Mechanism of the down-regulation of cAMP receptor protein by glucose in Escherichia coli: role of autoregulation of the crp gene.

Glucose causes catabolite repression by lowering the intracellular levels of both cAMP and cAMP receptor protein (CRP) in Escherichia coli. The molecular mechanism underlying the down-regulation of CRP by glucose has been investigated. We show that glucose lowers the level of crp mRNA without affecting its stability. Replacement of the crp promoter with the bla promoter almost completely abolishes the glucose-mediated regulation of crp expression. Only a slight reduction in the crp expression by glucose is observed in cya- or crp- strains, suggesting that a CRP-cAMP complex is needed for this regulation. We previously showed that transcription of the crp gene is regulated both negatively and positively. Positive autoregulation of crp is caused by the binding of CRP-cAMP to the CRP binding site II located upstream of the crp promoter. Here we show that disrupting the CRP binding site II essentially eliminates the down-regulation of crp expression by glucose. We conclude that the autoregulatory circuit of the crp gene plays a key role in the down-regulation of CRP by glucose.

Regulated tissue-specific expression of antagonistic pre-mRNA splicing factors.

The SR proteins are essential metazoan pre-mRNA splicing factors that can also influence the selection of alternative 5' splice sites in a concentration-dependent manner. Their activity in alternative splicing in vitro is antagonized by members of the hnRNP A/B family of proteins. The opposite effects of members of these two families of antagonistic splicing factors in vitro and upon overexpression in vivo suggest that changes in their relative levels may be a natural mechanism for the regulation of alternative splicing in vivo. One prediction of this model is that the ratios of these antagonists should vary in different cell types and in other situations in which cellular or viral transcripts are differentially spliced. We raised monoclonal antibodies specific for SF2/ASF and used them to measure the abundance of SF2/ASF protein and its isoforms, its phosphorylation state in vivo and during splicing in vitro, and its association with the spliceosome. SF2/ASF exists predominantly or exclusively in a highly phosphorylated state in vivo in all cell types examined, and unphosphorylated protein was not detectable. Unphosphorylated recombinant SF2/ASF becomes rapidly phosphorylated under splicing conditions in HeLa cell extracts and associates stably with one or more exons of beta-globin pre-mRNA. This interaction appears to persist through the splicing reaction and SF2/ASF remains bound to spliced mRNA. We compared the distribution of SF2/ASF to that of its antagonist, hnRNP A1, in different rat tissues and in immortal and transformed cell lines. We found that the protein levels of these antagonistic splicing factors vary naturally over a very wide range, supporting the notion that changes in the ratio of these proteins can affect alternative splicing of a variety of pre-mRNAs in vivo.

Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors.

The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by positive factors was investigated using two reconstituted mutant RNA polymerases (containing C-terminally truncated alpha subunits) and three positive factors [the cAMP receptor protein (CRP), OmpR, and PhoB]. The mutant RNA polymerases did not respond to transcription activation by activator proteins that bind upstream of the respective promoters. Transcription by these mutant enzymes was, however, activated in the cases where activators bind to target sites that overlap the promoter -35 region. Two different mechanisms are proposed for the positive control of transcription by activator proteins, one requiring the C-terminal domain of the alpha subunit, and the other not requiring it.