Minimal exon sequence requirements for efficient in vitro splicing of mono-intronic nuclear pre-mRNA

Measurements of the in vitro splicing efficiency of deletion mutant RNA precursors containing the small intron of the rabbit beta-globin gene, which are truncated in the first or in the second exon, revealed that no more than approximately 20 nucleotides of either exon are necessary for efficient splicing. At least for the second exon, this minimal length requirement is globin sequence-independent. Reduction of the exon-2 length to 14 nucleotides resulted in very inefficient splicing, whereas further reduction to 5 nucleotides apparently abolished the second splicing step (3' cutting and ligation), whereas the first step (5' cutting and branching) still occurred. The splicing efficiency of a double-mutant substrate retaining approximately 20 nucleotides of each exon was reduced to 50%. A kinetic study indicated that in the reaction of this double-mutant substrate the second, but not the first, splicing step was delayed, in contrast to the reaction of the wild-type precursor. Duplication or triplication of the entire sequence of exon-1 did not affect the splicing efficiency, whereas elongation of this exon with approximately 100 nucleotides of 5'-flanking (nontranscribed) beta-globin sequence diminished the level of correct splicing with the simultaneous appearance of aberrant lariat forms. We conclude that for mono-intronic precursors in which there is only one choice of splice sites, most of the exon sequences are not mechanistically involved in the splicing process.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Alzheimer's disease: study of the distribution of tau proteins constituting helical filament pairs in human central nervous tissue

Tau proteins are the major components of Paired Helical Filaments (PHF) of Alzheimer's disease. Using the immunoblot technique and an antiserum against PHF, we have studied the distribution of Tau proteins in the different areas of normal human brains and Alzheimer brains. Tau proteins were clearly present in cortical grey matter but were difficult to detect in the white matter. In Alzheimer brains, we observed two differences: first, there is an important background due to the partial dissociation of the lesions containing Tau aggregates. Second, the profile of Tau proteins is modified, due to abnormal phosphorylation. Thus, Tau proteins are found in large amounts in the grey matter of the cortical areas and are not exclusively distributed in the axonal domain. The normal cortical distribution of Tau in the human brain correlates well with the distribution of histological lesions that contain PHF (neurofibrillary tangles and neuritic plaques) in the Alzheimer cortex.     

A 2.6 kb intron separates the signal peptide coding sequence of an anther-specific protein from the rest of the gene in sunflower

Summary Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450_SU1 and P-450_SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450_SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15 kb of DNA, including the structural genes for cytochrome P-450_SU1 and an associated ferredoxin Fd-1 (suaC and suaB, respectively). In the absence of this monooxygenase system, the mutants described here respond to the presence of sulfonylureas or phenobarbital in the growth medium with the expression of only the suhC,B gene products (cytochrome P-450_SU2 and Fd-2), previously observed only as minor components in wild-type cells treated with sulfonylurea. These strains have enabled an analysis of sulfonylurea metabolism mediated by cytochrome P-450_SU2 in the absence of P-450_SU1, yielding an in vivo delineation of the roles of the two different cytochrome P-450 systems in herbicide metabolism by S. griseolus.     

Anther-specific,developmentally regulated expression of genes encoding a new class of proline-rich proteins in sunflower

We have used RNA gel blot analysis to demonstrate the anther-specific expression of three genes in sunflower. Expression of these genes was first detected shortly before flower opening, which occurs sequentially on the sunflower inflorescence, and continues during pollination. In contrast, these genes are not expressed (or only weakly expressed) in a male-sterile line in which anther development aborts. In situ hybridization experiments showed that these genes are only expressed in the single cell layer of the sunflower anther epidermis. In the case of one of these genes, which codes for an abundant mRNA, we report the peptide sequences deduced from the sequence of two similar but non identical cDNAs. These proteins contain a potential signal peptide and are characterized by the presence of a proline-rich region which reads KPSTPAPPPPPP(PP)K. Our results also suggest that several proline-rich proteins of unknown functions are specifically synthesized during the maturation of anthers in sunflower.     

Swainsonine treatment accelerates intracellular transport and secretion of glycoproteins in human hepatoma cells

We are interested in determining whether carbohydrates are important regulatory determinants in the intracellular transport and secretion of glycoproteins. In the present study, we have used swainsonine, an indolizidine alkaloid, to modify the structure of N-glycosidically linked complex oligosaccharides. By inhibiting Golgi mannosidase II, swainsonine prevents the trimming of GlcNAc(Man)5(GlcNAc)2 to GlcNAc-(Man)3(GlcNAc)2, resulting in the formation of hybrid-type oligosaccharides. We find, from pulse-chase experiments using [35S]methionine and immunoprecipitation of individual proteins from culture media, that swainsonine treatment (1 microgram/ml) accelerated the secretion of glycoproteins (transferrin, ceruloplasmin, alpha 2-macroglobulin, and alpha 1-antitrypsin) by decreasing the lag period by 10-15 min relative to untreated cultures. The enhanced secretion was specific for glycoproteins since the secretion of albumin, a nonglycoprotein, was unaffected. When alpha 1-antitrypsin was immunoprecipitated from the cell lysates, sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorographic analysis demonstrated that the conversion of the high-mannose precursor to the hybrid form in swainsonine-treated cells occurred more rapidly (by about 10 min) than the conversion to the complex form in control cells. Since both the hybrid and complex forms of alpha 1-antitrypsin are terminally sialylated by sialyltransferase in the trans-Golgi, these results suggest that swainsonine-modified glycoproteins traverse the Golgi more rapidly than their normal counterparts. Therefore, accelerated transport within this organelle may account for the decreased lag period of glycoprotein secretion in the swainsonine-treated cultures.     

Differentiation of the rat epididymis after withdrawal of androgen

Summary The differentiation capacity of the rat epididymis after depletion of androgen was studied in organ culture and in castrated rats. The differentiation of narrow cells in 5- and 10-day-old explants and in 10-day-old castrated rats suggests that: (i) the testicular androgens are not essential for their differentiation, (ii) a differential androgen dependence exists among the epididymal cell types, (iii) the undifferentiated epithelial cells are the precursors of the narrow cells.     

Differential effects of 1-deoxynojirimycin on the intracellular transport of secretory glycoproteins of human hepatoma cells in culture

To investigate our earlier hypothesis that carbohydrates play a regulatory role in the intracellular transport of secretory glycoproteins, we used 1-deoxynojirimycin (DNJ), and inhibitor of glucosidase I and II of the rough endoplasmic reticulum (RER), to modify the structure of N-linked glycan moieties of secretory glycoproteins of human hepatoma (Hep G2) cells in culture. Using a pulse-chase protocol, we found that treatment of Hep G2 cultures with 1.25 mM DNJ markedly reduced the rate of secretion of ₁-protease inhibitor, ceruloplasmin, and ₂-macroglobulin, but had no effect on the export of fibronectin, -fetoprotein and transferrin, nor on albumin which lacks carbohydrate. For example, 50% of newly synthesized ₁-protease inhibitor, the glycoprotein most dramatically affected, was secreted by 27 min in control cultures versus 110 min in DNJ-treated cultures. Percoll gradient cell fractionation analyses revealed that DNJ inhibited transport of the affected secretory glycoproteins in the RER segment of the ER/Golgi pathway. For example, 50% of newly synthesized ₁-protease inhibitor was lost from the RER fraction by 10 min in untreated cells, but 70 min was required for the transport of a similar amount of protein in DNJ-treated cells. DNJ treatment also inhibited the rate at which the N-linked glycan moieties of the affected glycoproteins became resistant to endo H in the Golgi. Since the glycan moiety of secreted forms of the affected glycoproteins were fully processed to the complex structure, suggesting escape from DNJ inhibition, we concluded that removal of terminal glucose residues from the glycan chain of secretory glycoproteins is required for their transport from the RER to the Golgi. We suggest that the oligosaccharide moieties on ₁-protease inhibitor, ceruloplasmin and ₂-macroglobulin form part of the binding site for a receptor which regulates transport of these glycoproteins.