During protein synthesis, the ribosome catalyzes peptide-bond formation. Biochemical and structural studies revealed that conserved nucleotides in the peptidyl-transferase center (PTC) and its proximity may play a key role in peptide-bond formation; the exact mechanism involved remains unclear. To more precisely define the functional importance of the highly conserved residues, we used a systematic genetic method, which we named SSER (systematic selection of functional sequences by enforced replacement), that allowed us to identify essential nucleotides for ribosomal function from randomized rRNA libraries in Escherichia coli cells. These libraries were constructed by complete randomization of the critical regions in and around the PTC. The selected variants contained natural rRNA sequences from other organisms and organelles as well as unnatural functional sequences; hence providing insights into the functional roles played by these essential bases and suggesting how the universal catalytic mechanism of peptide-bond formation could evolve in all living organisms. Our results highlight essential bases and interactions, which are shaping the PTC architecture and guiding the motions of the tRNA terminus from the A to the P site, found to be crucial not only for the formation of the peptide bond but also for nascent chain elongation.nucleotide essentiality ͉ protein biosynthesis ͉ symmetrical region R ibosomes are universally conserved ribonucleoproteins that translate the genetic information contained in mRNAs into proteins. The large (50S) ribosomal subunit catalyzes peptide-bond formation at the peptidyl-transferase center (PTC) between aminoacyl-tRNA (aa-tRNA) bound to the A site and peptidyl-tRNA (pep-tRNA) at the P site. In the crystal structures of 50S subunits of Haloarcula marismortui, H50S (1, 2), Deinococcus radiodurans, D50S (3, 4), and 70S ribosomes (5, 6), the PTC is composed solely of 23S rRNA and, hence, acts as a ribozyme, consistent with biochemical findings using deproteinized 50S subunit (7-10) for the formation of a single peptide bond.The PTC provides the frame for peptide-bond formation (11, 12) and plays a critical role in tRNA and nascent chain release (13-17), and the global ribosomal architecture is crucial for substrate positioning (18,19). Consistently, the hypothesis, based on structures of H50S complexed with minimum substrates, that the PTC acts as a general acid-base catalyst (2, 20-22), was contradicted by various mutagenesis and biochemical studies (12,(23)(24)(25)(26)(27)(28). The main catalytic contribution of the ribosomes, substrates positioning at proper orientation (4,28,29), is achieved by remote interactions, accompanied by symmetrical base-pairing of C75 of both tRNAs with G2553 (Escherichia coli numbering throughout) and G2251 (Fig. 4A, which is published as supporting information on the PNAS web site), respectively (2,4,31). The distinction between the rates of peptide-bond formation by full-length tRNAs and minimal substrates is also consistent with the essential role ...

Helix 38 (H38) in 23 S rRNA, which is known as the "A-site finger (ASF)," is located in the intersubunit space of the ribosomal 50 S subunit and, together with protein S13 in the 30 S subunit, it forms bridge B1a. It is known that throughout the decoding process, ASF interacts directly with the A-site tRNA. Bridge B1a becomes disrupted by the ratchet-like rotation of the 30 S subunit relative to the 50 S subunit. This occurs in association with elongation factor G (EF-G)-catalyzed translocation. To further characterize the functional role(s) of ASF, variants of Escherichia coli ribosomes with a shortened ASF were constructed. The E. coli strain bearing such ASF-shortened ribosomes had a normal growth rate but enhanced ؉1 frameshift activity. ASF-shortened ribosomes showed normal subunit association but higher activity in poly(U)-dependent polyphenylalanine synthesis than the wild type (WT) ribosome at limited EF-G concentrations. In contrast, other ribosome variants with shortened bridge-forming helices 34 and 68 showed weak subunit association and less efficient translational activity than the WT ribosome. Thus, the higher translational activity of ASF-shortened ribosomes is caused by the disruption of bridge B1a and is not due to weakened subunit association. Single round translocation analyses clearly demonstrated that the ASF-shortened ribosomes have higher translocation activity than the WT ribosome. These observations indicate that the intrinsic translocation activity of ribosomes is greater than that usually observed in the WT ribosome and that ASF is a functional attenuator for translocation that serves to maintain the reading frame.Ribosomes are universally conserved ribonucleoproteins that translate the genetic information contained in mRNAs into proteins. In bacteria, the large (50 S) and the small (30 S) subunits associate to form functional 70 S ribosomes. Both subunits are connected by 12 intersubunit bridges formed by RNA-

Ribosomes translate the genetic information contained in mRNAs into proteins. The large (50 S) subunit of the ribosome catalyzes the formation of a peptide bond between the aminoacyl-tRNA (aa-tRNA) 3 bound to the A-site and the peptidyl-tRNA at the P-site. This peptide bond formation takes place at the peptidyltransferase center of the 50 S subunit. Codon-anticodon pairing occurs at the decoding center of the small (30 S) subunit. The aa-tRNA is delivered to the ribosome as a ternary complex of aa-tRNA, EF-Tu, and GTP. Cognate codon recognition is strictly monitored by 16 S rRNA and triggers GTP hydrolysis and dissociation of EF-Tu. This allows aa-tRNA to be accommodated by the A site of the 50 S subunit. Thus, accuracy of protein synthesis is based on the synergistic interplay of the large and small subunits of the ribosome. However, mechanistic insights into the ribosome dynamics during decoding are still rudimentary.The intersubunit bridges of the ribosome are functional sites that are not only necessary for subunit connection but also play roles in translation. Helix 69 (H69) (position 1906 -1924) is a highly conserved stemloop in domain IV of 23 S rRNA of the bacterial 50 S subunit (Fig. 1A). In fact, each base in the loop of H69 shows more than 98% conservation in 436 bacterial rRNAs (www.rna.icmb.utexas.edu). Crystallographic studies revealed that H69 is located on the surface involved in intersubunit association with the 30 S subunit by connecting with helix 44 (h44) of 16 S rRNA forming the bridge B2a; A1912, A1913, A1914, A1918 and A1919 in H69 make contact with positions 1407-1410 and 1494 -1495 in h44 and G1517 in h45 (1, 2) (see Fig. 7A). In the free 50 S subunit, H69 makes a compact structure and interacts with H71 of 23 S rRNA (3), whereas in the 70 S ribosome H69 stretches toward the small subunit and interacts with h44 of 16 S rRNA (1). The tip of H69 moves about 13.5 Å during this structural change. In addition, H69 directly interacts with A/T-, A-, and P-site tRNAs during each translation step (1). During the decoding step, aa-tRNA is brought into the A/T-site as a complex with EF-Tu/GTP (ternary complex). Cryoelectron microscopy analyses showed a kinked conformation for aa-tRNA at the A/T state (molecular spring) (4, 5). The tip of H69 makes a contact with the hinge of the kink between D-and anticodon-stems in tRNA. This interaction is supposed to facilitate the structural distortion of tRNA that enables the anticodon-stem to fit into the decoding center of 16 S rRNA. In the crystal structure of the 70 S ribosome complexed to both A-and P-site tRNAs, H69 is positioned between the two tRNAs (1). The minor groove of H69 (positions 1908 -1909, 1922-1923) interacts with the minor groove of the D-stem of the P-site tRNA (positions 12-13, 25-26) (Fig. 1B), whereas the conserved loop (positions 1913-1915) of H69 makes a contact with the D-stem of A-site tRNA (positions 11-12 and 25-26) (Fig. 1B). Moreover, it has been reported that H69 interacts with various translational factors. In the post-t...

Recently, it has been shown that the homozygous deletion of the cyclin- dependent kinase-4 inhibitor (CDK4I;p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor-suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.

A new type of water-in-oil-in-water (W/O/W) emulsion using lipophilized gelatin (LG) and cotton seed oil was developed for the administration of salmon gonadotropin (sGtH) to induce ovarian matura tion in the Japanese eel. Hormone-releasing properties of the LG emulsion were compared with a W/O type emulsion prepared with Freund's incomplete adjuvant (FIA) and saline solution in in vitro and in vivo experiments. These results indicated that the LG emulsion had hormone-releasing properties differ ent from those of the saline solution and the FIA emulsion. Cultured immature female Japanese eel (BW, 566 to 1017 g) received a weekly intramuscular injection (total of 10 injections) of the LG emul sion, the FIA emulsion or the saline solution, each of which contained sGtH. In the group treated with LG emulsion containing sGtH, all fish matured showing small individual variations in GSI and percent increases in body weight. Although the FIA emulsion was also effective in inducing ovarian maturation, it is possible that ovarian maturation was inhibited by antibodies produced against sGtH. Administra tion of sGtH in saline solution required a longer period of time for the completion of ovarian matura tion. Therefore, it appeared that the LG emulsion containing sGtH was more effective than other prepa rations in inducing ovarian maturation in eel.

A patient with a squamous cell carcinoma accompanied by a marked granulocytosis ( 100,000/mm3) of unknown origin was examined for Colony-Stimulating Activity ( N PATIENTS WITH nonhematological malig-

SUMMARYA rapid test based on an immunochromatography assay -Determine Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine TM is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.

Eighty-one bronchoalveolar lavage (BAL) specimens obtained from 26 HIV-infected, 45 non-HIV immunosuppressed and 10 immunocompetent patients with primary pulmonary diseases were analysed for the presence of Pneumocystis carinii by staining and by P. carinii 5S rDNA determined by PCR. P. carinii was observed by staining of BAL specimens from HIV-infected patients significantly more frequently than those from immunocompromised hosts without HIV infection (57.7% versus 20.0%, respectively). P. carinii 5S rDNA was detected by PCR assay in seven (26.9%) HIV-infected individuals, which was significantly more frequent than for four (8.9%) immunosuppressed patients without HIV infection, for whom staining was negative. None of these patients developed P. carinii pneumonia (PCP) within the follow-up period. BAL specimens from 10 immunocompetent patients with pulmonary disorders were negative for PCP by both staining and PCR assay.