Protein folding in Escherichia coli: role of 23S ribosomal RNA.

Post-translational control of Escherichia coli ribosome on newly synthesised polypeptide leading to its active conformation (protein folding) has been shown in the case of the enzyme beta-galactosidase. As expected, antibiotics chloramphenicol and lincomycin, which bind to 23S rRNA/50S subunit and kasugamycin and streptomycin which interact with the 30S subunit instantaneously inhibited protein synthesis when they were added to the growing cells. The increase in beta-galactosidase activity, though stopped immediately after the addition of chloramphenicol and lincomycin, went on considerably in the presence of streptomycin and kasugamycin even after the stoppage of protein synthesis.

Reactivation of denatured proteins by domain V of bacterial 23S rRNA.

In vitro transcripts containing domain V of the 23S rRNA of Escherichia coli and Bacillus subtilis can reactivate denatured proteins almost as efficiently as the total 23S rRNA. Here we show that almost the full length of domain V is required for reactivation of denatured pig muscle lactate dehydrogenase and pig heart cytoplasmic malate dehydrogenase: the central loop of this domain alone is not enough for this purpose. The antibiotic chloramphenicol, which binds to domain V of 23S rRNA, can inhibit reactivation of these proteins completely. Activity is eliminated by EDTA at a concentration of <1 mM, even in the presence of 4 mM MgCl2, suggesting that the three-dimensional conformation of the RNA should be maintained for this activity.

Medulloblastoma--a retrospective analysis.

A retrospective review of 45 patients was undertaken at the All India Institute of Medical Sciences to assess the outcome and prognostic factors for these patients who received post operative radiotherapy with or without chemotherapy for medulloblastoma. The median age at diagnosis was 11 years, with 34 males and 11 female patients. Thirty four tumours were confined to midline structures, and 11 were localised to one cerebellar hemisphere or involved midline and lateral structures. Complete macroscopic removal was achieved in 24 patients and subtotal removal in 21 patients. Forty one patients underwent craniospinal irradiation and 27 patients received adjuvant chemotherapy. Median overall and disease free survival was 57 and 31 months respectively and 3 year overall survival was 76%. The addition of adjuvant chemotherapy was a significant factor for disease free survival (p = 0.01) whereas extent of surgery (total vs subtotal, p = 0.01) was a significant factor for overall survival only. Eleven patients developed recurrent disease, with ten relapsing first in the posterior fossa.

Purification and characterization of an endo-exonuclease from adult flies of Drosophila melanogaster.

An endo-exonuclease (designated nuclease III) has been purified to near homogeneity from adult flies of Drosophila melanogaster. The enzyme degrades single- and double-stranded DNA and RNA. It has a sedimentation co-efficient of 3.1S and a strokes radius of 27A The native form of the purified enzyme appears to be a monomer of 33,600 dalton. It has a pH optimum of 7-8.5 and requires Mg2+ or Mn2+ but not Ca2+ or Co2+ for its activity. The enzyme activity on double-stranded DNA was inhibited 50% by 30 mM NaCl, while its activity on single-stranded DNA required 100 mM NaCl for 50% inhibition. Under the latter conditions, its activity on double-stranded DNA was inhibited approximately 98%. The enzyme degrades DNA to complete acid soluble products which are a mixture of mono- and oligonucleotides with 5'-P and 3'-OH termini. Supercoiled DNA was converted by the enzyme to nicked and subsequently to linear forms in a stepwise fashion under the condition in which the enzyme works optimally on single-stranded DNA. The amino acid composition and amino acid sequencing of tryptic peptides from purified nuclease III is also reported.

Reactivation of denatured fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase with E. coli ribosome.

Fungal glucose 6-phosphate dehydrogenase and E. coli alkaline phosphatase were denatured either by physical or by chemical means. In vitro reconstitution of these denatured enzymes was assisted by 70S E. coli ribosome, as shown by the recovery of their catalytic competence. Almost total recovery of the activities of completely inactivated enzymes was obtained when 70S ribosome was present at about equimolar concentration with the enzyme molecules at 37C and 50C, respectively.

Isolation and characterization of the biotin genes of Escherichia coli K-12.

DNA containing the biotin gene cluster, bioABFCD, of E. coli K-12 has been isolated from the EcoRI cleavage products of lambdabiot124-10 phage DNA and subsequently characterized by electron microscopic studies. The biotin-DNA fragment obtained after EcoRI cleavage of the lambdabiot124-10 DNA measures 18.7% lambda DNA length (approx. 9000 base pairs). In addition to the biotin genes, it contains 4.75% and 3.08% lambda phage DNA at the left and right end-points of the bioABFCD cluster, respectively. The two bio promoter sites of the divergently transcribed biotin genes have been visualized under the electron microscope by binding RNA polymerase holoenzyme to the biotin DNA fragment. The two promoters are located at 41% and 43% length of the DNA fragment from its left endpoint. In vitro transcription of RNA from the bio-tin-DNA fragment has been visualized with the electron microscope, but so far no simultaneously transcribing "RNA:DNA" loops of the divergently oriented genes have been observed.