Processing of chloroplast ribosomal RNA transcripts in Euglena gracilis bacillaris

The ribosomal RNA operons ( rrn operons) of Euglena gracilis chloroplasts contain genes for (in order) 16S rRNA, tRNA Ile , tRNA Ala , 23S rRNA and 5S rRNA. Major sites of cleavage of the primary rrn transcript were identified by Northern blot hybridization and S1-mapping. The presumptive termini of all of the mature products have now been identified. During initial processing in the chloroplast, the primary transcript is cleaved between the two tRNAs and between the 23S and 5S rRNAs so as to separate the sequences found in the different mature rRNAs. Subsequently the tRNAs are separated from the rRNAs, further trimming provides the remaining proper ends, and the 3′-ends of the tRNAs are added.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46969/1/294_2004_Article_BF00419917.pd

Initiation of rrn transcription in chloroplasts of Euglena gracilis bacillaris

The site of initiation of chloroplast rRNA synthesis was determined by Sl-mapping and by sequencing primary rRNA transcripts specifically labeled at their 5′-end. Transcription initiates at a single site 53 nucleotides upstream of the 5'-end of the mature 16S rRNA under all growth conditions examined. The initiation site is within a DNA sequence that is highly homologous to and probably derived from a tRNA gene-region located elsewhere in the chloroplast genome. A nearly identical sequence (102 of 103 nucleotides) is present near the replication origin. The near identity of the two sequences suggests a common mode for control of transcription of the rRNA genes and initiation of chloroplast DNA replication. The related sequence in the tRNA gene-region does not appear to serve as a transcript initiation site.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46967/1/294_2004_Article_BF00521275.pd

Extracellular matrix proteases contribute to progression of pelvic organ prolapse in mice and humans

Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, fibulin-5 inhibited the β1 integrin–dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5RGE/RGE) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to fibulin-5 knockouts (Fbln5–/–), Fbln5RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with β-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5–/– mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre- and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women

Cloning and expression in Escherichia coli

Recombinant plasmids containing the mosquitocidal δ-endotoxin gene were constructed by inserting HindIII fragments of the Bacillus thuringiensis var. israelensis 72-75 Md plasmid in to the Escherichia coli vector pUC12. Two recombinants producing the 26000 Da δ-endotoxin (pIP173 and pIP174) were identified by screening clones in an E. coli in vitro transcription-translation system. Both recombinants were 12.4 kb chimaeric plasmids comprising pUC12 and a common 9.7 kb HindIII fragment of the B. thuringiensis plasmid. The 26000 Da polypeptide synthesis in vivo from pIP174 transformed into E. coli JM101 was lethal to mosquito larvae and cytotoxic to mosquito cells in vitro. The biological authenticity of the cloned product was further confirmed by demonstrating that the cytotoxicity of the polypeptide was neutralised by antiserum to the authentic δ-endotoxin or by preincubation with excess toxin receptor. Transcription of the recombinant δ-endotoxin gene in E. coli appears to utilise a Bacillus promotor sequence(s) rather than the pUC12 β-galactosidase promotor.</p