Transcription termination and RNA processing in the 3''-end spacer of mouse ribosomal RNA genes.

The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically.     

The effects of guanine and cytosine variation on dinucleotide frequency and amino acid composition in the human genome

Summary One hundred twelve human DNA sequences were analyzed with respect to dinucleotide frequency and amino acid composition. The variation in guanine and cytosine (G+C) content revealed: (1) at 2–3 and 3-1 doublet positions CG discrimination is attenuated at high G+C, but TA disfavor is enhanced, and (2) several amino acids are subject to G+C change. These findings have been reported in part for collections of sequences from various species. The present study confirms that in a single organism-the human-the G+C effects do exist. Aspects of the argument that connects G+C with protein thermal stability are also discussed.     

Immunocytochemical localization of cathepsins B, H, and their endogenous inhibitor, cystatin beta, in islet endocrine cells of rat pancreas

To determine the characteristics of lysosomes in rat islet endocrine cells, we examined the precise localization of cathepsins B, H, and L and their specific inhibitors, cystatins alpha and beta, using immunocytochemical techniques. By use of serial semi-thin sections, we detected immunoreactivity for cathepsin B in insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-positive (PP) cells. Strong immunoreactivity for cathepsin H was seen in A-cells and weak immunoreactivity in PP cells, but none in others. Immunodeposits for cystatin beta were demonstrated in B-cells. Brief dipping of thin sections in 1% sodium methoxide before the following immunocytochemical reaction enhanced specific deposits of immunogold particles on the target organelles. Use of a double-immunostaining technique showed co-localization of insulin with cystatin beta in many secretory granules. This suggests that cystatin beta may regulate converting enzymes participating in the maturation process of insulin. By use of an immunogold technique, heterogeneous localization of cathepsins B and H in lysosomes was also found among islet cells at the light microscopic level. This may be due to the difference in peptides degraded in lysosomes among the cells.     

Localization of gastrin-releasing peptide (GRP)-like immunoreactivity in the lysosomal compartment of the trigeminal ganglion cells of the rat

Summary Cellular and subcellular localizations of gastrin-releasing peptide-like immunoreactivity (GRP-LI) were examined in the perikarya of trigeminal ganglion cells. By immunolight microscopy using semi-thin sections, GRP-LI was observed in almost all the neuronal somata with various intensity as granular profiles distributing widely in the cytoplasm. By immunoelectron microscopy using ultrathin frozen sections and protein A-gold, GRP-LI was found predominantly in rounded or oval membrane-bound structures which were 300–800 nm in diameter and displayed various electron-density and heterogenous contents. Double-labeling immunoelectron microscopy using antibodies for GRP and cathepsin L, a well-characterized lyosomal proteinase, clearly demonstrated that these GRP-immunoreactive intracytoplasmic structures were lysosomes. In contrast, GRP-LI was detected only occasionally in the large granular vesicles (100–200 nm in diameter). These findings strongly suggest that considerable amount of GRP or GRP-like peptide is subject to intracellular degradation in the lysosome rather than to the regulatory secretion pathway, and this is the reason why no fibers immunoreactive for GRP have been detected in the peripheral sensory field.     

Effect of proteinase inhibitors on intracellular processing of cathepsin B, H and L in rat macrophages

The effects of various proteinase inhibitors on the processing of lysosomal cathepsins B, H and L were investigated in cultured rat peritoneal macrophages. The processing of newly synthesized pro-cathepsins B, H and L to the mature single-chain enzymes was sensitive to a metal chelator,1,10-phenanthroline, and a synthetic metalloendopeptidase substrate, Z-Gly-Leu-NH2, and insensitive to inhibitors of serine proteinases, aspartic proteinases and cysteine proteinases. Inhibitors of cysteine proteinases, E-64-d and leupeptin, inhibited the processing of the single-chain forms of cathepsins B, H and L to the two-chain forms. These results suggest that (a) metal endopeptidase(s) is (are) involved in the propeptide processing of cathepsin B, H and L, and that proteolytic cleavages of the mature single-chain cathepsins are accomplished by cysteine proteinases in lysosomes.