Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage.

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.

Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis.

A polyprotein composed of multiple units arranged in direct tandem arrays has been identified in parasitic and free living nematodes. Analysis of previously cloned units from the Dirofilaria immitis polyprotein antigen (DiPA) indicated the units were nearly identical but here we demonstrate that they segregate into two related families. The consensus repeats, DiPA-CR1 and CR2, derived for each family are 80% identical. However, the repeats at the C-terminus of the polyprotein have diverged from DiPA-CR1 and CR2. This was shown by DNA sequence and Southern blot analysis of a 1.9 kb cDNA clone that encodes 4.4 C-terminal repeats (DiPA-TR1 through TR5). DiPA-TR3 through TR5 show 27-52% amino acid identity with the consensus repeats and 31-35% amino acid identity with one another. Metabolic labeling studies have shown that cleavage of DiPA generates a protein "ladder' from 14 to > 200 kDa. RRKR, a cleavage motif of subtilisin-like proprotein convertases, was identified as the natural cleavage site. In vitro digestion experiments with proteinase K suggest a structural model for DiPA consisting of protease resistant cores joined by protease sensitive linkers containing the RRKR site. This motif is absent between DiPA-TR3 and TR4 and has been altered to KR between DiPA-TR4 and TR5. An immunoblot of D. immitis extract probed with anti-DiPA-TR4/5 serum demonstrates the absence of cleavage at these sites. These divergent repeats provide an opportunity to investigate processing of the D. immitis polyprotein in vivo.

A novel beta-galactosidase gene isolated from the bacterium Xanthomonas manihotis exhibits strong homology to several eukaryotic beta-galactosidases.

The gene encoding a beta-galactosidase from Xanthomonas manihotis was cloned into Escherichia coli. The gene resides on a 2.4 kb DNA fragment which was isolated from a partial Sau3A library in the cloning vector pUC19 using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as the selection. The enzyme produced by the clone has a specificity for beta 1-3- > beta 1-4-linked galactose. The nucleotide sequence of the gene was determined. The deduced protein sequence contained 597 amino acids yielding a monomeric molecular mass of 66 kDa. The cloned beta-galactosidase showed no similarity to any known prokaryotic beta-galactosidase. However, extensive similarity was observed with eukaryotic beta-galactosidases from animals, plants and fungi. The strongest similarity was with the beta-galactosidases found in the human and mouse lysosomes (42 and 41% identity, respectively). Alignment of the X.manihotis and eukaryotic beta-galactosidase sequences revealed seven highly conserved domains common to each protein. Additionally, Domain 1 in X.manihotis showed similarity to regions within catalytic domains from seven xylanases and cellulases belonging to family 10 of glucosyl hydrolases. A region spanning Domain 2 showed similarity to the catalytic domain of endo beta 1-3 glucanases from tobacco and barley.

Cloning and expression of the NaeI restriction endonuclease-encoding gene and sequence analysis of the NaeI restriction-modification system.

NaeI, a type-II restriction-modification (R-M) system from the bacterium Nocardia aerocolonigenes, recognizes the sequence 5'-GCCGGC. The NaeI DNA methyltransferase (MTase)-encoding gene, naeIM, had been cloned previously in Escherichia coli [Van Cott and Wilson, Gene 74 (1988) 55-59]. However, none of these clones expressed detectable levels of the restriction endonuclease (ENase). The absence of the intact ENase-encoding gene (naeIR) within the isolated MTase clones was confirmed by recloning the MTase clones into Streptomyces lividans. The complete NaeI system was finally cloned using E. coli AP1-200 [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] and less stringent MTase-selection conditions. The naeIR gene was expressed first by cloning into S. lividans, and later by cloning under control of a regulated promoter in an E. coli strain preprotected by the heterologous MspI MTase (M.MspI). The DNA sequence of the NaeI R-M system has been determined, analyzed and compared to previously sequenced R-M systems.

In vitro protein splicing of purified precursor and the identification of a branched intermediate.

Protein splicing is a posttranslational processing event in which an internal polypeptide is excised from a protein precursor and the terminal polypeptides are then ligated together, resulting in the production of two proteins. This report presents direct evidence for protein splicing by demonstrating in vitro splicing of purified precursor that accumulated when the protein splicing element from Pyrococcus DNA polymerase was cloned into a foreign gene. In vitro splicing was temperature and pH dependent. A slowly migrating species exhibited kinetic properties of a splicing intermediate and was shown to be a branched molecule by N-terminal sequencing. The precursor and slowly migrating species were interconvertible in response to pH shifts.

Characterization and expression of the Escherichia coli Mrr restriction system.

The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.

Lectin cytochemistry reveals differences between hamster trachea and bronchus in the composition of epithelial surface glycoconjugates and in the response of secretory cells to neutrophil elastase.

Hamsters exposed to an intratracheal instillation of human neutrophil elastase (HNE) accumulate an abnormally high number of secretory granules in bronchial but not tracheal epithelial cells. We employed lectin cytochemistry to investigate possible differences in the epithelial cell surface glycoconjugate layer in trachea compared to bronchus which might explain the regional dissimilarity in response to HNE. Portions of glutaraldehyde-fixed trachea and bronchi were incubated in one of several ferritin-labeled lectins prior to embedding for transmission electron microscopy. Lectins from Ricinus communis, Helix pomatia, and Triticum vulgaris bound to the surface of tracheal secretory cells in moderate to profuse amounts, while most bronchial secretory cells showed little or no label with these lectins. Gold-labeled Helix pomatia agglutinin (HPA), a lectin specific for secretory cells, showed a decrease in surface binding to all tracheal secretory cell types within 2 h of HNE instillation, compared to saline controls. In contrast, the majority of bronchial secretory cells showed an HNE-induced increase in surface label from extremely low levels in saline controls. The low levels of lectin binding to bronchial cells, in contrast to the trachea, may indicate the lack of a protective surface glycoconjugate coat, thus explaining the vulnerability of these cells to HNE. The rise in number of accessible HPA binding sites on the surface of bronchial secretory cells exposed to HNE may represent an important event in the pathologic accumulation of secretory granules by these cells.

The ultrastructure of hamster bronchial epithelium.

The central intrapulmonary bronchi of adult male Syrian hamsters were examined by electron microscopy to identify the principal types and proportions of epithelial cells. A differential count of cells displaying both a basal lamina and luminal border (transepithelial cells) showed that, on average, ciliated cells constituted 63% and granule-containing (granulated) secretory cells 25% of the total. Other transepithelial cells included nongranulated secretory cells (9%), preciliated cells (1.5%), and indeterminate cells (1%). The most frequent granulated secretory cell (77% of the population) was the Clara cell. It was identified by the presence of prominent apical smooth endoplasmic reticulum and secretory granules. It was subclassified into three types based on the presence or absence of rough endoplasmic reticulum and on granule morphology. Mucous cells (little or no smooth endoplasmic reticulum but with typical mucous granules) constituted approximately 20% of the granulated secretory cells. Serous cells were very infrequent. A differential count of nucleated epithelial cells demonstrated an average of 2% basal cells (hemidesmosomes present) and 20% pseudobasal cells (hemidesmosomes absent). Neuroepithelial bodies and solitary "small-granule" cells were infrequent. Brush cells and apoptotic bodies were rarely found but are noteworthy because their occurrence in hamster airways was not demonstrated previously. These results provide a foundation for subsequent analysis of alterations of epithelial homeostasis induced by injurious agents of exogenous and endogenous origin.

An ultrastructural study of the response of hamster bronchial epithelium to human neutrophil elastase.

The central intrapulmonary bronchi of hamsters were examined by transmission electron microscopy at varying times following intratracheal instillation of human neutrophil elastase (HNE) or its vehicle, saline. Two hours after HNE treatment, there was a marked irregularity of the surfaces of many nonciliated epithelial cells; a differential count of transepithelial cells (those with both a basal lamina and luminal border) demonstrated a significant decrease in the proportion of granule-containing (granulated) secretory cells and a corresponding increase in nongranulated secretory cells. By 3 days after HNE injection, the differential count had returned to control levels and cell surface alterations were less evident. By 8 days, the proportion of granulated secretory cells had significantly increased, while that of nongranulated secretory cells had decreased. Many Clara cells developed the characteristics of mucous cells so that mucous cells constituted 57% of the secretory cells compared to 14% for the saline controls. The mucous cells contained an increased number of mucous granules including bizarre forms never seen in controls. By day 16, the average mucous cell proportion had increased to 75%; the mucous cells were larger and contained many more secretory granules than at day 8. At no time was there evidence of overt cell injury or alteration of extracellular connective tissue due to HNE. Basal and pseudobasal cells, distinguished by the presence or absence of hemidesmosomes, did not change as a percentage of total nucleated epithelial cells. Saline had no effect on the differential cell count compared to untreated values. Our results indicate a strong likelihood that HNE causes early discharge of secretory granules and alters the phenotypic expression of Clara cells so that they produce abundant, often abnormal mucous granules. The mechanism of HNE-induced disturbance of epithelial homeostasis is unknown, but the early irregularity of nonciliated epithelial cell surfaces may signify an important event in the evolution of the resultant lesion.