Isolation of putative plant transcriptional coactivators using a modified two-hybrid system incorporating a GFP reporter gene.

Dual hybrid interacting screening in yeast led to the identification of two proteins from Arabidopsis both exhibiting sequence similarity to a family of transcriptional coactivators from a diverse range of organisms. Their discovery constitutes the first description of such plant proteins. A modified yeast two-hybrid approach utilising the green fluorescent protein (GFP) of Aequora victoria was developed and used to clone one of the putative plant transcriptional coactivators from an Arabidopsis cDNA library. The two proteins, designated KIWI and KELP, can associate both hetero- and homomerically and their genes were cloned and mapped on the Arabidopsis genome. Both proteins are believed to play a role in gene activation during pathogen defence and plant development. The involvement of these proteins in general plant transcription as well as the advantages of using GFP as a reporter gene for detecting protein-protein interactions are discussed.

Rapid amplification of genomic ends (RAGE) as a simple method to clone flanking genomic DNA.

This report describes the amplification of upstream genomic sequences using the polymerase chain reaction (PCR) based solely on downstream DNA information from a cDNA clone. In this novel and rapid technique, genomic DNA (gDNA) is first incubated with a restriction enzyme that recognizes a site within the 5' end of a gene, followed by denaturation and polyadenylation of its free 3' ends with terminal transferase. The modified gDNA is then used as template for PCR using a gene-specific primer complementary to a sequence in the 3' end of its cDNA and an anchored deoxyoligothymidine primer. A second round of PCR is then performed with a second, nested gene-specific primer and the anchor sequence primer. The resulting PCR product is cloned and its sequence determined. Three independent plant genomic clones were isolated using this method that exhibited complete sequence identity to their cDNAs and to the primers used in the amplification.

Increase in Mallampati score during pregnancy.

A photographic version of the Mallampati test was developed and applied to 242 pregnant patients at 12 weeks' gestation and again at 38 weeks' gestation. At 38 weeks the number of grade 4 cases had increased by 34% (P < 0.001). This is in agreement with other evidence which suggests that difficult laryngoscopy is slightly more frequent in obstetrics (1.7%) than in general surgery (1.3%). The increase in Mallampati score correlated with gain in body weight (r = 0.3, P < 0.001), which gives some support to the concept that fluid retention is the underlying cause. We conclude that pharyngeal oedema causes some hindrance to tracheal intubation in obstetrics, but not enough to explain the high failure rate reported. A case is made for rationalizing the management of difficult intubation. Our data also show that more research is needed on factors which affect Mallampati's test, particularly neck extension.

RNase E activity is conferred by a single polypeptide: overexpression, purification, and properties of the ams/rne/hmp1 gene product.

Ribonuclease E, an enzyme that processes pre-5S rRNA from its precursor, is now believed to be the major endoribonuclease participating in mRNA turnover in Escherichia coli. The product of the ams/rne/hmp1 gene, which is required for RNase E activity, was overexpressed, purified to near homogeneity by electroelution from an SDS/polyacrylamide gel, and renatured. The purified polypeptide possesses nucleolytic activity in vitro with a specificity identical to that observed for crude RNase E preparations. In addition, both UV crosslinking and RNA-protein blotting unambiguously showed that the Ams/Rne/Hmp1 polypeptide has a high affinity for RNA. Our results demonstrate that RNase E activity is directly attributable to, and is an inherent property of, an RNA-binding protein, the ams/rne/hmp1 gene product.

Structural requirements for the processing of Escherichia coli 5 S ribosomal RNA by RNase E in vitro.

Processing of 9 S precursor RNA in Escherichia coli requires the endoribonuclease RNase E, which makes two cleavages to liberate p5, the immature form of 5 S rRNA. The contributions of primary and secondary structure to RNase E-mediated cleavage of 9 S RNA were investigated. The structure of the 5' domain of 9 S RNA was probed by partial ribonuclease digestion and chemical modification. Our structural analysis of 9 S RNA supports a model in which the 5' spacer domain folds into tandem hairpins so that the first processing cleavage site 5' to the 5 S moiety resides in a stretch of single-stranded residues. Site-directed mutagenesis of a cloned 9 S RNA sequence was performed and synthetic transcripts derived from a variety of such mutant templates were assayed as substrates for RNase E-dependent endonuclease activity in fractionated extracts. Partial or complete deletion of the 5 S sequence did not eliminate site-specific processing of 9 S RNA. Mutations affecting the 5' domain revealed that secondary structure upstream from the first cleavage site is important in maintaining efficient processing. However, secondary structure downstream from either cleavage site is dispensable. Our results suggest that RNase E specifically recognizes and cleaves single-stranded RNA sequences only when presented in a proper conformational context. Adjacent secondary structures appear to play a direct and critical role in the enzyme's recognition of its substrate. Additionally, it may serve to anchor single-stranded regions to ensure the availability of the RNase E cleavage sites.

Mapping ribosomal protein S20-16 S rRNA interactions by mutagenesis.

Specific ribonucleotides within the 5' domain of Escherichia coli 16 S rRNA were altered by deletion and/or substitution by site-directed mutagenesis of cloned DNA to assess the importance of these particular residues in defining the binding site for ribosomal protein S20. Gel filtration and sucrose gradient centrifugation were employed to measure the binding of ribosomal protein S20 to synthetic transcripts spanning the 5' domain of 16 S rRNA containing various alterations. In several cases, the apparent association constants for these interactions were also determined. Three essential results were obtained. First, RNA transcripts containing residues 1-402 are sufficient for high affinity S20 binding. Second, bulges at residues 250-251 and 278-280 in a stem-loop structure extending from residues 240 to 286 are critical for S20 binding, arguing that the "260 stem" directly interacts with S20 at these sites. Third, mutations in a hairpin structure spanning residues 316-337 demonstrate a strong requirement for both the specific A321*G332 bulge and for unpaired residues in the loop itself for proper S20 binding. Our results document the importance of unpaired residues in maintaining the interaction between S20 and 16 S rRNA.

Unexpected, difficult laryngoscopy: a prospective survey in routine general surgery.

A prospective study of unexpected, difficult laryngoscopy was carried out. During a 7-month period, all general surgery patients in whom the trachea was intubated were assessed; only those with obvious neck pathology were excluded. Ease or difficulty of laryngoscopy was graded by a standard method. There were no grade 4 cases and no failed intubations in a total of 1387 cases. There were significant differences in the results recorded by different individuals; this did not correlate with seniority or with the type of surgery. Four factors have been identified which help to explain these discrepancies. These findings are analysed in relation to the training of junior staff, with particular reference to obstetric anaesthesia.