Evidence for an RNA binding region in the Escherichia coli processing endoribonuclease RNase E

The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA. A number of deletions were constructed in order to assess functional domains of the rne gene product. The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides. The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide. Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants. Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region. Binding to substrate RNAs correlated with the endonucleolytic activity. RNAs that are not substrates for RNase E did not bind to the protein. The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished. The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity.     

Phase II trial of paclitaxel and granulocyte colony-stimulating factor in patients with pancreatic carcinoma: a Southwest Oncology Group study

PURPOSE: Pancreatic cancer is difficult to treat, with most patients surgically unresectable at the time of diagnosis. Radiotherapy and chemotherapy can offer palliation, but more effective therapy is needed. This trial evaluated the effects of an aggressive schedule of paclitaxel given with granulocyte colony-stimulating factor (G-CSF) to patients with advanced pancreatic cancer. PATIENTS AND METHODS: All patients were required to have a histologic diagnosis of pancreatic adenocarcinoma with measurable disease and no prior chemotherapy or radiation therapy. Patients had to have performance status of 0 to 2, pretreatment absolute granulocyte count > or = 1,500/microL, and platelet count greater than or equal to the institutional lower limit of normal. Following pretreatment with dexamethasone, diphenhydramine, and cimetidine, patients received paclitaxel at a dose of 250 mg/m2 by 24-hour infusion on day 1, repeated every 21 days. G-CSF was given at a dose of 5 microg/kg/d on days 3 to 18 or until two consecutive absolute neutrophil counts (ANCs) > or = 10,000/microL were obtained. Doses of paclitaxel were modified depending on nadir counts. RESULTS: Forty-five patients were entered onto this study, with six ineligible. For the 39 eligible patients, there was one complete response (CR) and two partial responses (PRs), five stable/no responses, 23 increasing disease, two early deaths, and six patients whose assessment was inadequate to determine response. The response rate was therefore three of 39 or 8% (95% confidence interval [CI], 2% to 21%). The median survival time for the 39 eligible patients was 5 months. The most common toxicities were anemia, leukopenia/granulocytopenia, malaise/fatigue, nausea/vomiting, alopecia, thrombocytopenia, paresthesias, and liver function abnormalities. There was one death due to sepsis. CONCLUSION: Single-agent paclitaxel in this dose and schedule has minimal activity in pancreatic adenocarcinoma patients.     

Bryostatin 1 induces ubiquitin COOH-terminal hydrolase in acute lymphoblastic leukemia cells

It has been previously reported that Bryostatin 1 (Bryo1) induces differentiation of the human acute lymphoblastic leukemia (ALL) cell line, Reh, to a monocytoid B-cell stage. In this study we demonstrate that a novel protein, ubiquitin COOH-terminal hydrolase (UCH-L1), is associated with this differentiation. Reh cells were treated with 200 nmol/l of Bryo1 for 72 h and analyzed for changes in morphology, surface immunophenotype, acid phosphatase and terminal deoxynucleotidyl transferase. Protein patterns of the parent and differentiated cells, by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), were studied. Bryo1-treated cells expressed morphologic, phenotypic and enzymatic features of the monocytoid B-cell stage. The UCH-L1 enzyme (MW-pl 34-5.3) was detected by 2 D PAGE in the differentiated, but not in parent cells. The presence of UCH-L1 in the Bryo1-treated cells was further confirmed by immunoblotting of 2 D PAGE using UCH-L1 polyclonal antibody. Ubiquitin expression was studied in parent and Bryo1-treated cells and was compared with 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated cells. Both agents, TPA and Bryo1, increased the level of ubiquitin expression as detected by flow cytometry. Sodium borohydride, an inhibitor of UCH-L1, inhibited the Bryo1-induced differentiating effect on Reh cells. To date, the mechanism by which Bryo1, exerts its B-cell differentiating effect is not fully understood. This study shows that UCH-L1 expression may play a major role in Bryo1-induced differentiation in pre-B-ALL.     

Sequence diversity in the NIb coding region of eight sugarcane mosaic potyvirus isolates infecting sugarcane in Australia

We have sequenced the NIb coding region of sugarcane mosaic potyvirus strain SC (SCMV-SC) and eight field isolates of SCMV from Australia. This region comprised 1563 nucleotides and encoded a putative protein of 521 amino acids containing the consensus motif GDD. The protease cleavage sites between the NIa/NIb and the NIb/coat protein were found to be Q/C and Q/A, respectively. The SCMV sequences were most similar to sorghum mosaic potyvirus with identities of 70% and 78% at the nucleotide and amino acid levels, respectively. When the sequences were compared to each other, there was a maximum of 3.3% variation between isolates at the nucleotide level and a maximum of 0.8% at the amino acid level. Phylogenetic analysis of the sequences indicated the field isolates were grouped according to their geographical location. The SCMV sequence with most homology to all other isolates has been selected to generate constructs for replicase-mediated resistance.     

A longitudinal study of green-liver osteomyelitis complex in commercial turkeys

Two flocks of Nicholas tom turkeys from separate farms with histories of above-average condemnations for turkey green-liver osteomyelitis complex (TOC) were studied throughout a 16-week growout. Fifty birds from each farm were necropsied each week for 15 weeks, and birds that had green livers, osteomyelitis in the proximal tibia, or swollen joints were cultured for aerobic bacteria along with an equal number of control birds. At processing, TOC lesions and green livers were obtained for bacterial culture and histopathology. Green-liver-associated TOC was not observed until the turkeys were 9 or 10 weeks of age. The incidence of TOC was higher on one farm, which also had a higher incidence of airsacculitis, higher early and weekly mortality, seroconversion to Newcastle disease virus and Mycoplasma meleagridis, and significantly higher average body weights, relative spleen weights, and relative liver weights. Both farms had a high incidence of intestinal lesions and infestation with Ascaridia dissimilis. Histological evaluation of green livers revealed hyperplasia of bile ducts, dilation of sinusoids, and pigment-containing Kupffer's cells, some of which stained positive for iron. The bacterial isolates most frequently cultured from bones and livers were pleomorphic gram-variable coccobacilli, which grew visible colonies only after a series of subcultures and extended incubation.     

Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5' splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.