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Biomedical subjects

D Leclerc

Publications and source records attributed to D Leclerc.

At least 55 records · Page 3Linked to original sources
PubMed1992

Efficiency of the 5'-terminal sequence (omega) of tobacco mosaic virus RNA for the initiation of eukaryotic gene translation in Escherichia coli.

Recent studies have demonstrated that the 5' leader (omega sequence) of tobacco mosaic virus RNA has a certain enhancing capacity for translation of mRNA in both prokaryotes and eukaryotes. In order to estimate the efficiency of omega to initiate translation of mRNA in Escherichia coli, in comparison to the Shine-Dalgarno (S/D) sequence, we have inserted eight different eukaryotic genes into two types of E. coli expression vectors containing one constitutive promoter (P1) but different translation-initiation sites (S/D or omega delta 3 sequence, respectively). The efficiency of transcription and translation in vivo was evaluated for these vectors by measuring the yield of protein and both the level and stability of mRNA. We report that substitution of omega delta 3 for S/D decreases the yield of expressed protein 4-1900-fold and the content of gene-specific mRNA is decreased by about sevenfold. However, in comparison with the S/D sequence, the level of protein expressed under the translational control of omega delta 3 is less sensitive to changes in the 5' coding region. We also report that the omega sequence contains a region of 10-12 nucleotides complementary to the small ribosomal subunit RNA (rRNA) of E. coli, Eikenella corrodens and Xenopus laevis, and to the rRNA of the (small ribosomal) subunit of Oryza sativa.

Amino Acid Sequence↗
PubMed1992

Left ventricular pseudoaneurysm associated with mitral regurgitation.

A patient with a large posterolateral left ventricular pseudoaneurysm flush with the posterior papillary muscle and associated with mitral regurgitation was successfully treated surgically. The operation consisted of opening the sac of the aneurysm, dissecting the wall of the left ventricle from the fibrous wall of the sac, leaving a cuff of fibrous tissue suitable to hold sutures, and closing the defect with a Dacron patch.

Echocardiography↗
PubMed1991

Mutations in the 915 region of Escherichia coli 16S ribosomal RNA reduce the binding of streptomycin to the ribosome.

The nine possible single-base substitutions were produced at positions 913 to 915 of the 16S ribosomal RNA of Escherichia coli, a region known to be protected by streptomycin [Moazed, D. and Noller, H.F. (1987) Nature, 327, 389-394]. When the mutations were introduced into the expression vector pKK3535, only two of them (913A----G and 915A----G) permitted recovery of viable transformants. Ribosomes were isolated from the transformed bacteria and were assayed for their response to streptomycin in poly(U)- and MS2 RNA-directed assays. They were resistant to the stimulation of misreading and to the inhibition of protein synthesis by streptomycin, and this correlated with a decreased binding of the drug. These results therefore demonstrate that, in line with the footprinting studies of Moazed and Noller, mutations in the 915 region alter the interaction between the ribosome and streptomycin.

Bacterial Proteins↗
PubMed1991

A conformational switch involving the 915 region of Escherichia coli 16 S ribosomal RNA.

A novel alternative conformation, which involves an interaction between the 5' terminal and 915 regions (E. coli numbering), is proposed after a screening of compiled sequences of small subunit ribosomal RNAs. This conformation contains a pseudoknot helix between residues 12-16 and 911-915, and its formation requires the partial melting of the 5' terminal helix and the disruption of the 17-19/916-918 pseudoknot helix of the classical 16 S rRNA secondary structure. The alternate pseudoknot helix is proximal to the binding site of streptomycin and various mutations in rRNA which confer resistance to streptomycin have been located in each strand of the proposed helix. It is suggested that the presence of streptomycin favours the shift towards the alternate conformation, thereby stabilizing drug binding. Mutations which destabilize the novel pseudoknot helix would restrict the response to streptomycin.

Base Sequence↗
PubMed1991

Granulomatous renal mass during endovesical BCG therapy for bladder carcinoma. Diagnosis by fine-needle aspiration.

One case of pseudotumoral granulomatous renal mass during endovesical bacillus Calmette-Guérin (BCG) therapy for superficial bladder neoplasm is reported. Such an adverse effect is exceptional and is clearly related to a vesicorenal reflux in our patient. In this case ultrasound-guided fine-needle aspiration was able to settle the diagnosis and avoid surgery. The patient responded to triple antituberculous therapy.

BCG Vaccine↗
PubMed1991

The interaction between streptomycin and ribosomal RNA.

The present study shows that a mutation in the 530 loop of 16S rRNA impairs the binding of streptomycin to the bacterial ribosome, thereby restricting the misreading effect of the drug. Previous reports demonstrated that proteins S4, S5 and S12 as well as the 915 region of 16S rRNA are involved in the binding of streptomycin, and indicated that the drug not only interacts with the 30S subunit but also with the 50S subunit. The relationship between the target of streptomycin and its known interference with the proofreading control of translational accuracy is examined in light of these results.

Autoradiography↗
PubMed1990

A deletion mutation at the 5' end of Escherichia coli 16S ribosomal RNA.

A deletion of five nucleotides was introduced at the 5' end of the Escherichia coli 16S rRNA gene cloned in an appropriate vector under control of a T7 promoter. The 16S rRNA generated by in vitro transcription could be assembled into 30S subunits. The deletion did not affect the efficiency of translation of natural messengers and the correct selection of the reading frame. However, it reduced the binding of the messengers, which suggests that the 5' end of 16S rRNA is located on the pathway followed by the messengers on the 30S subunits. The deletion also restricted the stimulation of misreading by streptomycin in a poly(U)-directed system. This is in accord with the proximity of the 5' end of 16S rRNA to proteins S4, S5 and S12, which are known to be involved in the control of translational accuracy.

Base Sequence↗
PubMed1990

The anti-Shine-Dalgarno region in Escherichia coli 16S ribosomal RNA is not essential for the correct selection of translational starts.

Plasmid pPM114, which contains the Escherichia coli 16S rRNA gene under control of a T7 promoter, was linearized upstream of the 3' end of the gene and used in an in vitro transcription assay to yield a 16S rRNA lacking about 30 nucleotides at its 3' end. This truncated 16S rRNA was assembled into 30S subunits which contain the full complement of 30S proteins, including S21, but were impaired in their capacity to associate to the 50S subunits. This impairment was paralleled by a decrease in their protein synthesis activity under the direction of natural or artificial messengers. However, although the anti-Shine-Dalgarno sequence was missing, the initiation step was not specifically affected, and the mutated ribosomes could initiate translation at the correct start sites. This supports previous suggestions that the translational efficiency and the selection of translational starts are not solely controlled by the Shine-Dalgarno interaction. A novel interpretation of the role of protein S21 is also proposed which is independent of the activation by this protein of the base-pairing potential of the anti-Shine-Dalgarno sequence of 16S rRNA.

Base Sequence↗
PubMed1990

Study of the function of Escherichia coli ribosomal RNA through site-directed mutagenesis.

Various approaches have been developed to study how mutations in Escherichia coli ribosomal RNA affect the function of the ribosome. Most of them are in vivo approaches, where mutations are introduced in a specialized plasmid harboring the ribosomal RNA genes. The mutated plasmids are then expressed in an appropriate host, where they can confer resistance to antibiotics whose target is the ribosome. Conditions can be used where the host ribosomal RNA genes or the host ribosomes are selectively inactivated, and the effect of the mutations on ribosome assembly and function can be studied. Another approach, which has been developed mainly with 16S ribosomal RNA, can be used entirely in vitro. In this approach, a plasmid has been constructed which contains the 16S ribosomal RNA gene under control of a T7 promoter. Mutations can be introduced in the 16S ribosomal RNA sequence and the mutated 16S ribosomal RNAs are produced by in vitro transcription. It is then possible to investigate how the mutations affect the assembly of the 16S ribosomal RNA into 30S subunits and the activity of the reconstituted 30S subunits in cell-free protein synthesis assays. Although these approaches are recent, they have already provided a large body of interesting information, relating specific RNA sequences to interactions with ribosomal proteins, to ribosome function, and to its response to antibiotics.

Base Sequence↗
PubMed1989

The conserved 900 stem/loop region in Escherichia coli 16S ribosomal RNA is not required for protein synthesis.

Plasmid pPM114 carries the Escherichia coli 16S ribosomal RNA gene under the control of a T7 promoter. It can generate in vitro transcribed 16S rRNA that can be assembled into functional 30S ribosomal subunits. Two deletion mutants were derived from pPM114, by partial or total deletion of the conserved 900 stem/loop region of the 16S rRNA. These mutants, pMG delta 10 and pMG delta 23, respectively lack bases 895 to 904 and 889 to 911 of the 16S rRNA. The amputated 16S rRNA transcripts synthesized from these mutated plasmids were assembled into 30S subunits which were as active under the direction of an artificial or a natural messenger as subunits reconstructed with the full-length 16S rRNA transcript. They also responded as well to the stimulation of misreading by streptomycin, although the deleted region is proximal to the streptomycin binding domain. However, when we attempted to delete the 895-904 or 889-911 region from the 16S rRNA gene in plasmid pKK3535 which carries the rrnB operon, no transformants harbouring plasmids with one of these deletions could be recovered. These observations suggest that the 900 stem/loop region of the 16S rRNA is not required for the ribosomal function but is probably essential for important cell regulatory functions.

Bacterial Proteins↗
PubMed1989

Lateralization of headache: possible role of an upper cervical trigger point.

An ipsilateral upper neck trigger point was found in 21 of 24 patients with unilateral headache. During the prodromic period this trigger point was detected as a tender protrusion on neck palpation. In 18 out of 24 patients it was also found during the headache-free period. On standard roentgenogram, this protrusion seemed to be a laterally developed C2 spinous process. The EMG study showed latent trapezius hypertonicity on the side of the headache, even during the headache-free period. The association of the painful protrusion and trapezius hypertonicity could create an autoreinforcing nociceptive loop, which in turn could be the cause of lateralization of the pain.

Adult↗
PubMed1989

Detection of bacterial cell wall hydrolases after denaturing polyacrylamide gel electrophoresis.

Cell walls from various Gram-positive bacteria were incorporated at a concentration of 0.2% (w/v) into polyacrylamide gels as a substrate for detection of cell wall hydrolases. Bacterial extracts from crude cell wall preparations were denatured with sodium dodecyl sulfate and 2-mercaptoethanol and subjected to denaturing polyacrylamide gel electrophoresis in gels containing bacterial cell walls. After renaturation in the presence of purified and buffered 1% (v/v) Triton X-100, cell wall hydrolases were visualized as clear lytic zones against the opaque cell wall background. One to fifteen bands with lytic activity could be detected, depending on bacterial extracts and on the nature of the cell walls incorporated into gels. Crude cell wall extracts were the best source of cell wall hydrolases from various Gram-positive bacteria such as Clostridium perfringens (15 bands), Micrococcus luteus (1 band), Bacillus megaterium (4 bands), Bacillus sp. (6 bands), B. cereus (3 bands), B. subtilis (7 bands), Staphylococcus aureus (13 bands), Streptococcus faecalis (3 bands), and Strep. pyogenes (5 bands). Molecular masses of cell wall hydrolases ranged from 17 to 114.6 kDa. Lytic activities against cell walls of Corynebacterium sepedonicum (Clavibacter michiganense pv. sepedonicum) could be shown with the cell wall extracts of Strep. pyogenes (45.7 kDa), Strep. faecalis (67 kDa), B. megaterium (67 kDa), and Staph. aureus (67 kDa).

Cell Wall↗
PubMed1988

Cloning, sequencing and expression of a Bacillus bacteriolytic enzyme in Escherichia coli.

Several hundred bacterial isolates were screened for bacteriolytic activity by growing them on agar medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells as the substrate. A Bacillus sp. producing the largest lytic zone was selected. A genomic bank of this selected bacterium was constructed in the multi-functional vector pTZ18R, with partial SauIIIA DNA fragments inserted at the SalI restriction site. Screening of 800 colonies of this bank for cell lysis gave 5 recombinants exhibiting lytic activity, as detected by analysis of extracts of sonicated Escherichia coli cells on denaturing polyacrylamide gels containing autoclaved, lyophilized M. lysodeikticus cells as the substrate. One clone (pBH2500), expressed in E. coli strain NM522, was found to code for a lytic enzyme corresponding, in molecular weight, to the 27 kDa Bacillus sp. hydrolase. This clone with an insertion of 2.5 kb was then subcloned as a 929 bp EcoRI-SauIIIA fragment in pTZ18R (pBH929) and showed higher cell lytic activity. A unique open reading frame for a protein of 251 amino acids, followed by a putative terminator sequence, was found after a consensus ribosome binding site. A putative leader sequence was identified in the first 37 amino acids. One truncated subclone (pBH703), corresponding to 196 out of 251 residues from the protein N-terminal end, still possessed lytic activity.

Amino Acid Sequence↗