Shigeo Masaki

Structural study of immunoaffinity-purified DNA polymerase α-DNA primase complex from calf thymus

The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.

Altered aggregation properties of mutant γ-crystallins cause inherited cataract

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non‐neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered γ‐crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of γ‐crystallin mutation, large inclusions containing the altered γ‐crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid‐detecting dye, Congo red. In vitro, recombinant mutant γB‐crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild‐type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid‐like inclusions. The mutant γ‐crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.

Knockout of the intermediate filament protein CP49 destabilises the lens fibre cell cytoskeleton and decreases lens optical quality, but does not induce cataract

In this report, the phenotype associated with the first targeted knockout of the lens specific intermediate filament gene CP49 is described. Several surprising observations have been made. The first was that no cataract was observed despite the fact that the beaded filaments of the lens fibre cells had been disrupted. Light scatter and the lens optical properties had, however, deteriorated in the CP49 knockout lenses compared to litter mate controls. These changes were accompanied by dramatic changes in plasma membrane organisation of the fibre cells as revealed by detailed morphological examinations and providing the second surprising result. The CP49 knockout mouse is therefore an important model to study the functional link between lens transparency, the cytoskeleton and plasma membrane organisation.

Detection of proliferative cells in dysplasia, carcinoma in situ, and invasive carcinoma of the uterine cervix by monoclonal antibody against DNA polymerase α

The distribution of DNA polymerase α‐positive cells in neoplasia of the uterine cervix and in normal cervical epithelium was studied using a monoclonal antibody against DNA polymerase α. The positive cells were found only in the parabasal layer of normal cervical epithelium and only in the nonkeratinized areas of the cancer nests of invasive keratinizing carcinoma. Most cells in cancer nests of an invasive nonkeratinizing carcinoma were found to be DNA polymerase α‐positive. In cases of mild or moderate dysplasia DNA polymerase α‐positive cells were found only in the lower half of the epithelium. DNA polymerase α‐positive cells in severe dysplasia to carcinoma in situ were distributed throughout the full thickness of the epithelium. The percentages of DNA polymerase α‐positive cells in mild or moderate dysplasia, severe dysplasia to carcinoma in situ, and invasive carcinoma were 32.2%, 45.7%, and 53.7%, respectively. The authors previously developed immunohistochemical methods for detecting DNA polymerase α by monoclonal antibody that allowed the proliferative activity of cells in normal and neoplastic tissues to be estimated.

cDNA sequence analysis of CP94: Rat lens fiber cell beaded-filament structural protein shows homology to cytokeratins

To study the molecular structure of the gene responsible for a lens fiber cell beaded-filament structural protein of 94kDa (CP94), we isolated its specific cDNA from a rat lens cDNA library by use of anti-mouse CP94 antiserum. The expressed fusion protein kept the epitopes specific against anti-chick CP97 as well as anti-mouse CP94 antibody, and the size was estimated as 190-200kDa, indicating that the cDNA insert of the clone seemed to encode a polypeptide with 80-90kDa in appearance. Northern analysis indicated that CP94 mRNA is expressed only in the lens, and not in the brain, skin, heart, kidney, lung, and liver, and the size was estimated to 2.1-2.3kb. In a lens of inherited microphthalmic mouse, Elo, a trace amount of mRNA with the size closely similar to that of rat mRNA was observed. The entire compiled sequence (1,873bp) showed an open reading frame covering the sequence of 533 amino acids totalling 58,857Da. No sequence homologous to the entire CP94 was found among the entries of any nucleotide and amino acid sequence databases; but with respect to a limited amino acid sequence of N-side region of CP94, a significant homology with cytokeratins was found.

Possible involvement of myosin-X in intercellular adhesion: Importance of serial pleckstrin homology regions for intracellular localization

Subcellular fractionation experiments with mouse hepatocytes, combined with sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE)–immunoblot analysis using antibodies against two different tail regions of mouse myosin‐X demonstrated a 240 kDa molecular mass to be associated with the plasma membrane‐rich P2 fraction. The basolateral plasma membrane fraction, but not the brush border fraction, isolated from renal cortices also contained the 240 kDa form of myosin‐X. In an attempt to assess relative contributions of possible functional domains in the tail of myosin‐X to localization and function, cDNA corresponding to all three pleckstrin homology (PH) domains and different regions (PH1, 2 and 3, and the two subdomains of PH1: PHS1 and PHS2), as well as the myosin tail homology 4 domain (MyTH4) and the band4.1/ezrin/radixin/moesin‐like domain (FERM) were separately inserted into the pEGFP vector and expressed in cultured COS‐1 cells. As a result, two distinct regions responsible for localization were identified with regard to PH: one covers all three forms that tends to localize to regions of dynamic actin, such as membrane ruffles, lamellipodia and thick cortical actin bundles at the sites of cell–cell adhesion in a Rac‐ and Cdc42‐dependent manner. The other covers PHS1 and PH2 that localizes to filopodia, filopodial puncta and the sites of intercellular adhesion in a Cdc42‐dependent manner. Expression of green fluorescent protein (GFP)‐MyTH4 fusion protein resulted in formation of phalloidin‐positive granules, while GFP‐FERM affected the actin cytoskeletal system in a distinctly different way. Taken altogether, the results lend support to the view that myosin‐X is involved in cell–cell adhesion‐associated signaling‐linked membrane and/or cytoskeleton reorganization.

Gene structure and sequence comparisons of the eye lens specific protein, filensin, from rat and mouse: implications for protein classification and assembly

The full length cDNA sequences of rat and mouse filensin are presented, as well as the structure of the rat filensin gene. This gene spanned 31 kb and included seven introns. The first six introns were conserved in position and phase with those found in the intermediate filament (IF) protein genes of the type II (type II keratin), type III (vimentin) and type V (lamin). The last intron of the filensin was unique. As none of the filensin intron positions coincided with those unique to type I, II or IV genes, it appears that filensin is most similar to type III genes. Comparison of the deduced amino acid sequences for rat and mouse filensin with those of cow and chick, and with other species of IF proteins, indicated the C-terminal non-alpha-helical tail domain of filensin to be one of the most divergent yet found in the vertebrate IF family. The tail domain had three conserved regions which are interrupted with two regions with lower identity. Two motifs, (1) PGDVPDGxxISKAF; and (2) KVEVVESIEKxxxxxIQTYEETxxIVET, were identified as sequences which were particularly highly conserved across species. Coassembly studies using CP49 and a physiologically derived 53 kDa-fragment of filensin showed the motif (2) was not required for filament assembly in vitro. These data strengthen the view that the C-terminal non-alpha-helical domain of filensin contributes in more than one way to filensin function in the lens.

DNA primase associated with 10 S DNA polymerase α from calf thymus

Abstract Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d 2 TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.

Defect of a fiber cell-specific 94-kDa protein in the lens of inherited microphthalmic mutant mouse Elo

Deficiency in a 94,000-dalton protein in the non-crystallin fraction from the Elo mouse lens was shown. To perform further investigations, we raised an antibody against the 94,000-dalton protein isolated from normal mouse lens. Western blot analysis with the antibody indicated that the protein was only present in the lens and not in the brain, lung, heart, liver, and kidney. In the lens, it was unique to the cortex and nucleus fractions, not being present in the epithelial cells. Furthermore, it was observed in the water-soluble fraction as well as in the urea-soluble fraction. The antibody weakly but clearly reacted with the chick CP97 lens peptide, a fiber cell-specific protein, and anti-CP97 antibody also reacted with the 94,000-dalton protein. From these results, we concluded that the protein corresponds to CP97 cytoskeletal protein in the mouse lens. The protein was deficient in the lenses from Elo mice, but microphthalmic lenses from CTA mice contained a normal level.

Linkage analysis of the mutation locus in the eye lens obsolescence (Elo) mouse.

To map precisely the mutation locus of eye lens obsolescence (Elo) on mouse chromosome 1, subsequent linkage analysis was achieved using backcross mating between 129/SvSl-Elo (Elo/+) and 129/SvSl (+/+). Mouse genomic DNAs from 17 strains including the Elo mutant mouse were first digested with several restriction enzymes and analyzed by hybridization using gamma 2- and gamma 4-crystallin cDNAs as probes. Restriction endonuclease DraI showed distinct RFLP patterns in both cases. When gamma 2-crystallin cDNA was used as the probe, two strong bands were observed at 4.0 and 2.4 kb in the majority of strains, but the former fragment shifted to the 3.4-kb position in 129/SvSl-Elo (Elo/Elo) and CFO. The polymorphism between 4.0- and 3.4-kb fragments corresponded to the gamma 1-crystallin locus (Cryg-1), and that of the 2.4-kb one, to the gamma 2-crystallin locus (Cryg-2). Mouse DNAs were also analyzed by hybridization using gamma 4-crystallin cDNA (Cryg-4). In this case, 3.4- and 3.0-kb fragments were observed in Elo and wild-type mice, respectively. The backcross offsprings were analyzed with respect to Elo, Idh-1, Cryg-1, and Cryg-4 loci. Among 223 mice analyzed, recombination between Elo and Idh-1 loci was observed in three offsprings; and that between Cryg-1 or Cryg-4 and Idh-1 loci, in one offspring. No recombination occurred between Cryg-1 and Cryg-4 alleles.