Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases

The RsrI endonuclease, a type-II restriction endonuclease (ENase) found in Rhodobacter sphaeroides, is an isoschizomer of the EcoRI ENase. A clone containing an 11-kb BamHI fragment was isolated from an R. sphaeroides genomic DNA library by hybridization with synthetic oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequence of RsrI. Extracts of E. coli containing a subclone of the 11-kb fragment display RsrI activity. Nucleotide sequence analysis reveals an 831-bp open reading frame encoding a polypeptide of 277 aa. A 50% identity exists within a 266-aa overlap between the deduced aa sequences of RsrI and EcoRI. Regions of 75-100% aa sequence identity correspond to key structural and functional regions of EcoRI. The type-II ENases have many common properties, and a common origin might have been expected. Nevertheless, this is the first demonstration of aa sequence similarity between ENases produced by different organisms.     

Phosphorylation of the peripherin 58-kDa neuronal intermediate filament protein. Regulation by nerve growth factor and other agents

Peripherin, a recently described member of the intermediate filament multigene family, is present in peripheral and certain central nervous system neurons as well as in cultured neuron-like cell lines, including PC12 pheochromocytoma cells. In PC12 cells, peripherin appears to be the major intermediate filament protein and its relative levels and synthesis are specifically increased during nerve growth factor (NGF)-promoted neuronal differentiation. The present study examines the phosphorylation of peripherin and the regulation thereof by nerve growth factor and other agents in cultured PC12 cells. Immunoblotting experiments using a peripherin-specific antiserum show five distinct isoforms of this protein in whole cell and cytoskeletal extracts resolved by two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of these isoforms incorporate detectable quantities of [32P]phosphate during metabolic radiolabeling. The small proportion (approximately 6%) of total cellular peripherin that is extractable with 1% Triton X-100, does not appear to incorporate phosphate. NGF increases peripherin phosphorylation by 2-3-fold within 1-2 h of treatment. Epidermal growth factor and insulin have no effect. The relative levels of phosphorylated peripherin are markedly elevated (17-fold) by long term NGF exposure, and peripherin becomes a major cytoskeletal phosphoprotein. Activators of protein kinases A and C and treatment with depolarizing levels of K+ also enhance peripherin phosphorylation by 2-3-fold, in cultures both with and without prior long term NGF treatment. Evidence is presented that NGF regulates peripherin phosphorylation by a mechanism independent of protein kinases A and C and of depolarization. The large increase in phosphorylated peripherin brought about by NGF treatment suggests that this neuronal filament protein may play a role in the elaboration and maintenance of neurites. The presence of multiple independent pathways that acutely enhance peripherin phosphorylation indicates that this role is subject to modulation by extrinsic signals.     

Molecular genetic study of human arginase deficiency

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.