The MDM‐2 (murine double minute 2) gene codes for a cellular protein that can bind to the p53 tumor suppressor gene product, thereby functioning as a negative regulator of p53. In order to define the role of the MDM‐2 gene in the pathogenesis of human acute myeloid leukemia, the expression and the sequence of the MDM‐2 gene were examined in samples of bone marrow and/or peripheral mononuclear cells of 38 patients by using immunostaining, polymerase chain reaction (PCR), single strand conformation polymorphism, and sequencing. Immunohistochemical staining detected a weak accumulation of the MDM‐2 protein in AML patients of FAB classification M4 and M5. RT‐PCR analysis revealed a heterogeneous expression pattern of MDM‐2 mRNA in AML samples of different FAB classification. An increased level of MDM‐2 mRNA expression was observed in 17 of 38 AML patients when compared to normal controls. No structural changes in a 488 bp region extending from nucleotide 890 to 1378 of the MDM‐2 cDNA were detected using RT‐SSCP and sequence analysis. In addition, heterogeneous expression of p53 transcripts was found with the highest p53 mRNA levels in AML M4 and M5. Interestingly, there seems to be a correlation between the relative ratios of p53 and MDM‐2 mRNA levels in AML M4 and M5: in 15 of 23 cases high p53 mRNA expression was directly associated with high levels of MDM‐2 transcripts. An exclusively intranuclear p53 immunostaining pattern was found in 10 of 16 (58%) AML FAB M4 and M5, whereas the remaining AML samples tested were negative for p53 (0/10). Using RT‐SSCP analysis and direct sequencing of the RT‐PCR amplification products of p53 exon 5–8, we observed that only 1 of 38 AML patients showed a point mutation in the p53 gene. This missense mutation occurred in the evolutionary highly conserved region of p53 at codon 255 (Ile to Phe). These data indicated that structural alterations of the p53 gene do not play an important role in the initiation and progression of AML. However, abrogation of p53 tumor suppressor function due to MDM‐2 overexpression may be an alternative molecular mechanism by which a subset of AMLs may escape from p53‐regulated growth control.

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From the gram-negative eubacterium Pseudomonas aeruginosa we have isolated a stable 6S RNA, approximately 180 nucleotides in length. The RNA was partially sequenced and identified by comparison with the known Escherichia coli 6S RNA sequence. Southern hybridizations revealed a single copy gene coding for the 6S RNA. DNA from other prokaryotes, i.e. E. coli, Thermus thermophilus, Bacillus subtilis, Bacillus stearothermophilus and Halobacterium maris mortui, did not give detectable hybridization signals. The 6S RNA gene was cloned in E. coli and its complete primary structure was determined. Although the 6S RNA sequences from P. aeruginosa and E. coli share only a 60.4% homology, we are able to propose a common secondary structural model.

Bacillus stearothermophilus large ribosomal subunits were reconstituted in the presence of 5S rRNAs from different origins and tested for their biological activities. The results obtained have shown that eubacterial and archaebacterial 5S rRNAs can easily substitute for B. stearothermophilus 5S rRNA in the reconstitution, while eukaryotic 5S rRNAs yield ribosomal subunits with reduced biological activities. From our results we propose an interaction between nucleotides 42-47 of 5S rRNA and nucleotides 2603-2608 of 23S rRNA during the assembly of the 50S ribosomal subunit. Other experiments with eukaryotic 5.8S rRNAs reveal, if at all, a very low incorporation of these RNA species into the reconstituted ribosomes.

Reconstitution experiments with 50 S ribosomal subunits from Bacillus stearothermophilus demonstrate that spinach chloroplast 5 S rRNA can be incorporated into the bacterial ribosome and yield biologically active particles, thereby establishing the eubacterial nature of chloroplast 5 S rRNA. In contrast, mitochondria from Locusta migratoria or bovine liver do not appear to contain discrete, low-M, RNAs, which can replace 5 S rRNA in the functional reconstitution of B. stearothermophilus ribosomes.Chloroplast 5 S rRNA Mitochondrial5 S rRNA 50 S ribosome Reconstitution Protein synthesis