Nucleotide sequence of threonine tRNA from Bacillus subtilis.

A threonine tRNA was purified from Bacillus subtilis W168 by a combined use of column chromatographic systems. The nucleotide sequence was determined to be pG-C-C-G-G-U-G-U-A-G-C-U-C-A-A-U-D-G-G-D(U)-A-G-A-G-C-A-A-C-U-G-A-C-U-mo5U-G-U-t6A-A-psi-C-A-G-U-A-G-m7G-U-U-G-G-G-G-G-T-psi-C-A-A-G-U-C-C-U-C-U-U-G-C-C-G-G-C-A-C-C-AOH, where about 40 % of D20 remained unmodified as U20. It consists of 76 nucleotides including a new minor nucleoside, 5-methoxyuridine (mo5U), which occupies the wobble position of anticodon.     

Nucleotide sequence of initiator tRNA from Mycobacterium smegmatis.

The nucleotide sequence of initiator tRNA from Mycobacterium smegmatis was determined to be pCGCGGGGUGGAGCAGCUCGGDAGCUCGCUGGGCUCAUAACCCAGAGm7GUCG CAGGU psi CGm1AAUCCUGUCCCCGCUACCAOH . The nucleotide sequence of Mycobacterium initiator tRNA was found to be the same as that of Streptomyces initiator tRNA, except that G46 and A57 were replaced by m7G46 and G57 , respectively. The striking feature of Mycobacterium initiator tRNA is the absence of ribothymidine at residue 54, and the presence of 1-methyladenosine at residue 58 which makes the sequence of this tRNA similar to that of eukaryotic initiator tRNA.     

Nucleotide sequence analysis of endogenous murine leukemia virus-related proviral clones reveals primer-binding sites for glutamine tRNA.

Nucleotide sequences of the region that corresponds to the site of tRNA primer binding for a functional retrovirus were determined in five murine leukemia virus-related sequence clones from mouse chromosomal DNA, which contain a unique 170 to 200-base-pair additional internal segment in the long terminal repeats. The 3'-terminal 18-nucleotide sequence of a major glutamine tRNA isoacceptor was found to match well with the putative primer binding site: 18 of 18 in three clones, 17 of 18 in one clone, and 16 of 18 in one clone. This implies that most of these endogenous proviral sequences of the mouse genome, if replicated as retroviruses, will be different from ecotropic murine leukemia viruses and most mammalian type C retroviruses in using glutamine tRNA, rather than proline tRNA, as a primer.     

Histochemical quantitation of gamma-glutamyl transferase in human leukemia cell lines.

The enzyme gamma-glutamyl transferase (gamma-GT) is involved in many biochemical systems, including the signal transduction of hematopoietic growth factors. Standard colorimetric gamma-GT assays require larger cell numbers than may be obtainable in many cases, such as with highly purified stem-cell populations. To study gamma-GT expression in limited populations, we used a histochemical stain to analyze gamma-GT semiquantitatively in cells of hematopoietic origin. Several human leukemic cell lines, including one with inducible increases in gamma-GT, were stained for gamma-GT and graded 0 through 4+ for the amount of positive granules. The gamma-GT activity demonstrated by this stain was found to be directly proportional to the gamma-GT activity obtained with a colorimetric assay and could be used to calculate approximate gamma-GT activity. This stain therefore provides a useful method for determining gamma-GT activity when limited cell numbers are available.     

Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples

In diagnostic research challenges, quantitative real-time PCR (QPCR) has been widely utilized in gene expression analysis because of its sensitivity, accuracy, reproducibility, and most importantly, quantitativeness. Real-time PCR base kits are wildly applicable in cancer signaling pathways, especially in cancer investigations. T-cell acute lymphoblastic leukemia (T-ALL) is a type of leukemia that is more common in older children and teenagers. Deregulation of the Notch signaling pathway promotes proliferation and inhibits apoptosis of the lymphoblastic T cells. The aim of this study was to investigate the effect of Notch signaling activation on the expression of target genes using real-time QPCR and further use this method in clinical examination after validation. Two T-ALL cell lines, Jurkat and Molt-4, were used as models for activation of the Notch signaling via over-expression of the Notch1 intracellular domain. Expression analysis was performed for six downstream target genes (NCSTN, APH1, PSEN1, ADAM17, NOTCH1 and C-MYC) which play critical roles in the Notch signaling pathway. The results showed significant difference in the expression of target genes in the deregulated Notch signaling pathway. These results were also verified in 12 clinical samples bearing over-expression of the Notch signaling pathway. Identification of such downstream Notch target genes, which have not been studied inclusively, provides insights into the mechanisms of the Notch function in T cell leukemia, and may help identify novel diagnoses and therapeutic targets in acute lymphoblastic leukemia.     

Nucleotide sequence of human placenta cytoplasmic initiator tRNA

Cytoplasmic initiator tRNA from human placenta has been purified. The nucleotide sequence of this tRNA has been determined and found identical to that of initiator tRNA from mammalian cytoplasm.     

Nucleotide sequence of a lysine tRNA from Bacillus subtilis.

A lysine tRNA (tRNA1Lys) was purified from Bacillus subtilis W168 by a consecutive use of several column chromatographic systems. The nucleotide sequence was determined to be pG-A-G-C-C-A-U-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-U-C-U-G-A-C-U-U(U*)-U-U-K-A-psi-C-A-G-A-G-G-m7G(G)-U-C-G-A-A-G-G-T-psi-C-G-A-G-U-C-C-U-U-C-A-U-G-G-C-U-C-A-C-C-AOH, where K and U* are unidentified nucleosides. The nucleosides of U34 and m7G46 were partially substituted with U* and G, respectively. The binding ability of lysyl-tRNA1Lys to Escherichia coli ribosomes was stimulated with ApApA as well as ApApG.     

Nucleotide sequence of cytoplasmic initiator tRNA from Tetrahymena thermophila.

The total primary structure of cytoplasmic initiator tRNA from Tetrahymena thermophila mating type IV, was determined by post labeling techniques. The sequence is pa-G-C-A-G-G-G-U-m1G-G-C-G-A-A-A-D-Gm-G-A-A-U-C-G-C-G-U-Psi-G-G-G-C-U-C-A-U-t6A -A-C-Psi-C-A-A-A-A-m7G-U-m5C-A-G-A-G-G-A-Psi-C-G-m1A-A-A-C-C-U-C-U-C-U-C-U-G-C- U-A-C-C-AOH. The nucleotide residue in the position next to the 5'-end of the anticodon of this tRNA (residue No. 33) is uridine instead of cytidine, which has been found in cytoplasmic initiator tRNAs from multicellular eukaryotic organisms. The sequence of three consecutive G-C base pairs in the anticodon stem common to all other cytoplasmic initiator tRNAs is disrupted in this tRNA; namely, the cytidine at residue 40 in this region is replaced by pseudouridine in Tetrahymena initiator tRNA.     

Cellular resistance against troxacitabine in human cell lines and pediatric patient acute myeloid leukemia blast cells

Troxacitabine is a cytotoxic deoxycytidine analogue with an unnatural L-configuration, which is activated by deoxycytidine kinase (dCK). The configuration is responsible for differences in the uptake and metabolism of troxacitabine compared to other deoxynucleoside analogues. To determine whether troxacitabine has an advantage over other nucleoside analogues several cell lines resistant to cladribine and gemcitabine were exposed to troxacitabine, while blast cells from pediatric leukemia patients were tested for cross-resistance with other deoxynucleoside analogues. The gemcitabine resistant AG6000 (IC50: >3000 nM), and the cladribine resistant CEM (IC50: 150 nM) and HL-60 (IC50: >3000 nM) cell lines, all with no or decreased dCK expression, were less sensitive to troxacitabine than their wild type counterparts (IC50; A2780: 410, CEM: 71 and HL-60: 158 nM). dCK protein expression in CEM was higher than in HL-60, which, in turn, was higher than in A2780. Catalytically inactive p53 seems to increase the sensitivity to troxacitabine. The patient samples showed a large range of sensitivity to troxacitabine, similar to other deoxynucleoside analogues. Cross-resistance with all other deoxynucleoside analogues was observed.     

Nucleotide sequence of starfish initiator tRNA.

The nucleotide sequence of starfish ovary initiator tRNA was determined to be pA-G-C-A-G-A-G-U-m1G-m2G-C-G-C-A-G-U-G-G-A-A-G-C-G-U-G-C-U-G-G-G-C-C-C-A-U-t6A-A-C-C-C-A-G-A-G-m7G-D-m5C-C-G-A-G-G-A-psi-C-G-m1A-A-A-C-C-U-C-G-C-U-C-U-G-C-U-A-C-C-AOH. The sequence was determined by a combination of the two different post-labeling techniques. Two-dimensional cellulose thin-layer chromatography was adopted for analysis of 5'-terminal nucleotides of tRNA fragments produced by formamide treatment. The nucleotide sequence of starfish initiator tRNA is very similar to that of mammalian cytoplasmic initiator tRNAs, but has seven different nucleotide residues and two modifications: residue 55 is psi instead of U, and residue 26 is unmodified G instead of m2G.