Isolation and purification of the reaction center (RC) and the core (RC-LH1) complex from Rhodobium marinum: the LH1 ring of the detergent-solubilized core complex contains 32 bacteriochlorophylls.

The reaction center (RC) and the core (RC-LH1) complex were isolated and purified from Rhodobium marinum; together with the LH1 complex [Meckenstock et al. (1992a) FEBS Lett. 311: 128], a complete set of RC, LH1 and RC-LH1 from the same wild-type strain of a purple photosynthetic bacterium can therefore now be made. Comparison of the BChl a/BPhe a ratio (determined by HPLC) between the RC and the RC-LH1 complexes lead us to the determination of the number of BChls in the LH1 ring to be 32.06+/-2.90, indicating that the LH1 ring from Rh. marinum consists of 16 alphabeta subunits.

Molecular cloning and sequencing of cDNAs encoding homologues of human Ku70 and Ku80 autoantigen from Xenopus and their expression in various Xenopus tissues.

We isolated cDNA clones encoding Ku70 and Ku80 homologues of Xenopus laevis from a cDNA library prepared from Xenopus oocytes. The nucleotide sequences of these Ku70 and Ku80 homologues have coding sequences of 1833 bp and a 611 aa protein, and 2178 bp and a 726 aa protein, respectively. The amino acid sequences deduced from the open reading frame of the Ku70 and Ku80 cDNA clones were highly homologous to those from Ku genes previously isolated, such as human (ca. 65% and ca. 62% identity, respectively) and mouse (ca. 65% and ca. 60%), and show a certain degree of homology to Drosophila (ca. 27% with Ku70), Caenorhabditis elegans (ca. 20% with Ku80) and Saccharomyces cerevisiae (ca. 23% and ca. 19%). Our detailed comparison of the predicted amino acid sequences among these species revealed the highly conserved octa-peptide LPFXXDIR common to both Xenopus Ku70 and Ku80 homologues in the region showing the high homology throughout the species tested. A Northern analysis using specific cDNA probes showed that Ku poly(A)+ mRNAs are expressed at high levels in Xenopus adult oocyte and testis.

Generation of triplet and cation-radical bacteriochlorophyll a in carotenoidless LH1 and LH2 antenna complexes from Rhodobacter sphaeroides.

The LH1 antenna complex and a native form of the LH2 complex were isolated from the carotenoidless R26 and R26.1 mutants of Rhodobacter sphaeroides by the use of a new detergent, sucrose monocholate. One-color, pump-and-probe transient Raman spectroscopy of these complexes using 351 nm, approximately 50 ps pulses showed the generation of the triplet state of bacteriochlorophyll a (BChl a), whereas measurements using 355 nm, approximately 12 ns pulses showed the generation of BChl a cation radical. Subpicosecond to nanosecond time-resolved absorption spectroscopy using 388 nm, 200 fs pulses for excitation showed rapid (<1 ps) generation of the triplet state and fast decay (<10 ps) of the singlet state of BChl a. Microsecond absorption spectroscopy confirmed the generation of BChl a cation radical. EPR spectroscopy using 532 nm, approximately 5 ns pulses for excitation established the generation of BChl a cation radical. The EPR line width suggested that the unpaired electron is shared by two BChl a molecules. In LH1, the yield of BChl a cation radical per complex was estimated to be about 80% of that in the reaction center, and in LH2 about 50%. Thus, rapid generation of the triplet state, and its subsequent transformation into the cation-radical state of BChl a have been shown to be intrinsic properties of B870 and B850 BChl a assembly in the carotenoidless LH1 and LH2 antenna complexes. In the case of the carotenoid-containing LH2 complex, the triplet states of BChl a and carotenoid (spheroidene) were generated immediately after excitation, but the triplet-state BChl a was quenched efficiently by the carotenoid so that no BChl a cation radical was generated. Thus, the photoprotective function of the carotenoid in this antenna complex is shown.

Aphidicolin induces alterations in Golgi complex and disorganization of microtubules of HeLa cells upon long-term administration.

Treatment of HeLa cells with aphidicolin at 5 or 0.5 microg/ml induced cell cycle arrest at G1/S or G2/M phase, respectively, and was accompanied by unbalanced cell growth. Long-term administration of aphidicolin (more than 48 h) resulted in noticeable loss of reproductive capacity though cells were viable at the time of treatment. Immunofluorescence with anti-Golgi membrane protein monoclonal antibody (mAbG3A5) showed disfigurement of the characteristic mesh-like configuration when cells were treated for more than 48 h. Interestingly, we found that the fragmented Golgi complex formed a ring around the nucleus in more than 20% of the cells. Immunoelectron microscopy using mAbG3A5 antibody demonstrated that the stack structure of the fragmented Golgi complex in aphidicolin-arrested cells appeared partially broken up and seemed to have converted to a vesicle-like structure. Analysis using an antibody to tubulin and anticentrosome human autoimmune serum showed that alterations in the Golgi complex were induced even by the lower 0.5 microg/ml dose. These alterations were accompanied by both changes in the distribution of microtubules and an increase in the number of centrosomes. These cells lost their distinct perinuclear microtubule organizing center (MTOC). On the other hand, treatment with aphidicolin at 5 microg/ml did not induce multiplication of the centrosome although the loss of distinct MTOC was still evident. No changes took place in the Golgi complex, microtubule, or centrosome of cells treated with 0.5 microg/ml aphidicolin when cycloheximide was added simultaneously to the culture.

Clinical manifestations due to a point mutation of the mitochondrial tRNAleu(UUR) gene in five families with diabetes mellitus.

It has been shown that an adenine (A) to guanine (G) transition at position 3243 of the mitochondrial transfer RNA(tRNA)leu(UUR) gene is associated with a subgroup of diabetes mellitus. Therefore, we screened for this transition in 86 patients with non-insulin-dependent diabetes mellitus (NIDDM) in which two or three generations were affected with diabetes, in 14 patients with insulin-dependent diabetes mellitus, and in 9 families with diabetes mellitus and/or associated disorders suggesting mitochondrial gene abnormalities. We failed to identify the mutation in 100 diabetic patients, 86 NIDDM and 14 insulin-dependent diabetes mellitus (IDDM). Out of the latter 9 families, we identified an A to G transition in 14 individuals in 5 families. Diabetes mellitus was shown to be maternally inherited in one family. In 9 of 14 patients with the mutation, insulin was required to treat diabetes mellitus, indicating impaired insulin secretion. A hyperglycemic clamp test performed in one subject revealed significant impairment of insulin secretion, whereas euglycemic clamp test showed normal insulin sensitivity in this patient. The heteroplasmy of the mutant mitochondrial DNA (mtDNA) in leukocytes does not appear to correlate with the severity of diabetes in terms of the insulin therapy required. Body mass index of the affected individuals was less than 23.3. In one family, in addition to diabetes mellitus and hearing loss, hypoparathyroidism was associated with the mutation, suggesting that hypoparathyroidism is caused by the impaired processing and/or secretion of proparathyroid hormone due to the mutation. In addition, the affected subjects presented with proteinuria at the time of diagnosis of diabetes mellitus which appeared not to be related with diabetic nephropathy.

Inhibition of nuclear envelope reconstitution in Xenopus interphase egg extract by hemin.

Addition of hemin to the nuclear reconstitution system of Xenopus interphase egg extract using sperm head chromatin resulted in abnormal pseudonuclei exhibiting flattened membrane patches randomly distributed both on the surface and inside the nuclei. The structures that resembled nuclear pores were observed on these flattened membrane patch structures. Although the nucleosome structure was formed as revealed by the micrococcal nuclease digestion, the B-type lamin uptake into the nuclei was inhibited by hemin. Using heminagarose affinity chromatography, we isolated several hemin-binding proteins from fully reconstituted pseudonuclei. Some of the hemin-binding proteins bound concanavalin A (Con A). Comparison of hemin-binding proteins with those isolated from both fractions of supernatant and pellet separated by high speed centrifugation of the egg extract showed that the hemin-binding proteins of pseudonuclei were supplied from both fractions. The uptake of nuclear hemin-binding proteins did not occur in the incompletely reconstituted nuclei resulting from addition of excess sperm chromatin to the system. These results suggest that the hemin-binding proteins participate in the late steps of nuclear reconstitution during formation of the nuclear envelope.

Process of dispersion and fragmentation of Golgi complex by microtubule bundles formed in taxol treated HeLa cells.

By means of a monoclonal antibody (mAbG3A5) against Golgi membrane glycoprotein, we have visualized the relative position of cytoplasmic polymerized microtubule bundles to Golgi stack cisternae in taxol treated HeLa cells, and found extensive fragmentation of the Golgi stack cisternae brought about by microtubule bundles. Within a 1 h period of taxol treatment, polymerization of cytoplasmic microtubules increased rapidly to form microtubule bundles, while the Golgi complex dispersed slightly along with the polymerized microtubule bundles. After 2 to 3 h taxol treatment the dispersal of the Golgi complex from the microtubule-organizing center (MTOC) to cytoplasmic periphery rapidly progressed in the direction which the microtubules ran. At early dispersal, the microtubule bundles were oriented apart from the stretched end of the Golgi stack cisternae and exhibited little direct contact with the Golgi stack cisternae membrane. The Golgi stack cisternae then began to wind around the microtubule bundles, followed by the beginning of fragmentation of the Golgi stack cisternae. At this step, some of the microtubules seemed to attach to a part of the Golgi stack cisternae. After prolonged exposure of cells to taxol (25 h) the microtubule bundles were highly developed throughout the cells and most of the Golgi fragments were trapped at their termini. In these cells, extensive fragmentation of Golgi stack cisternae occurred, resulting in small Golgi vesicles bound to the microtubule bundle.

Lack of mutations of preproparathyroid hormone gene in three kindreds with familial isolated hypoparathyroidism.

The mutations of the preproparathyroid hormone (preproPTH) gene have been reported to cause some cases of familial isolated hypoparathyroidism (FIH). We investigated the preproPTH gene of five affected subjects of three Japanese kindreds with FIH. The mode of inheritance in FIH of two families was thought to be autosomal dominant, and the FIH of the other was probably inherited in an autosomal recessive manner. Exons 1, 2 and 3 of the preproPTH gene and its exon-intron boundaries were analyzed with either polymerase chain reaction and single strand conformational polymorphism, or direct sequencing of the amplified DNA. We did not detect any mutations in the amplified regions of the preproPTH gene, but an A to G transition in intron 1 was identified in all of the affected subjects. Among them, four were heterozygote, and the other was homozygote. This transition was considered to be a polymorphism, which was the same as reported previously. These results indicate that the preproPTH gene abnormalities are not responsible for FIH in these families. Further studies are required to elucidate whether genes coding for other molecules, such as calcium-sensing receptor, are involved in FIH.

Three novel mutations of thyroid hormone receptor beta gene in unrelated patients with resistance to thyroid hormone: two mutations of the same codon (H435L and H435Q) produce separate subtypes of resistance.

We report three novel mutations of the thyroid hormone receptor beta (TR beta) gene in three unrelated Japanese patients with resistance to thyroid hormone (RTH). Patients A and B exhibited generalized resistance phenotype, while patient C displayed more pituitary-selective unresponsiveness. Direct sequencing of TR beta gene exon 10 disclosed novel point mutations in all three patients. A Phe to Ile (TTC-->ATC) substitution at codon 451, a His to Leu (CAT-->CTT) substitution at codon 435, and a His to Gln (CAT-->CAA) substitution at codon 435 were identified in patients A, B, and C, respectively. Sequencing of TR beta gene exons 5-9 as well as TR alpha gene exons 4-9 failed to detect any additional mutations. All three patients were heterozygous for respective mutations. The unaffected parents of patients A and B, having normal thyroid function, possessed no mutations of TR beta gene exon 10, indicating that the F451I and H435L mutations occurred de novo. The F451I mutation is located near the most frequent mutation site in the ligand 2 subdomain. The identical codon mutations H435L and H435Q, which lie at the extreme carboxyl-terminus of the dimerization subdomain near the 9th heptad, were found in clinically different subtypes of RTH: patient B with generalized resistance and patient C with pituitary-selective resistance, respectively. The mutations broaden the growing catalogue of the TR beta gene mutations that could cause different phenotypes, despite the defects at the same codon.

Golgi membrane vesicles in HeLa mitotic cells are identified with monoclonal antibody made against Golgi cisternal membrane protein p138.

A monoclonal antibody (mAbG3A5) recognizing p138 antigen was used to identify the Golgi cisternal membrane and determine behavior of Golgi fragments during mitosis in HeLa cells. At the start of mitosis, Golgi stacks identified with the mAbG3A5 antibody were fragmented into fine membrane vesicles which were distributed throughout the cytoplasm leaving only the region of the chromosome cluster unoccupied. On Western immunoblotting analysis, p138 was found associated with the membrane fraction prepared from mitotic HeLa cells having a buoyant density the same as that of interphase Golgi membranes. In addition to the fine membrane vesicles, clusters labeled with mAbG3A5 antibody were frequently observed in mitotic cells. They numbered 11 on average per mitotic cell and consisted of fine membrane vesicles of which membrane region was labeled with the mAbG3A5 antibody. This fact indicates that the membrane vesicles in mitotic Golgi clusters were also part of the fragments of Golgi cisternae. The number of mitotic Golgi clusters per mitotic cell was constant from prophase to anaphase, increasing twofold at telophase, although the average size of mitotic Golgi cluster remained unchanged throughout mitosis. The increase in number of mitotic Golgi clusters at telophase was accompanied by decrease in immunofluorescence of fine membrane vesicles. Treatment with nocodazole caused the disappearance of the mitotic Golgi clusters from prophase cells; however upon removal of it, they were reformed. These results suggest that during mitosis the Golgi apparatus were fragmented to fine membrane vesicles leaving only a part as mitotic Golgi clusters and were reassembled through tentative clustering of the fine membrane vesicles at the end of mitosis.

Aphidicolin-sensitive DNA polymerase is incorporated into the chromatin during nuclear envelope assembly in Xenopus egg extract.

The mechanism for incorporation of aphidicolin-sensitive DNA polymerase into reconstituting sperm nuclei was studied in a Xenopus egg extract cell-free system. Aphidicolin-sensitive DNA polymerase activity was sedimented along with the light membrane fraction of Xenopus egg extract on a discontinuous sucrose gradient. Treatment of the egg extract with Triton X-100 caused DNA polymerase activity to migrate to a lighter density position at which free proteins were distributed. DNA polymerase activity was incorporated into the reconstituting sperm nuclei from the egg extract, but no nuclear incorporation was observed in nuclei incubated in egg extracts which had been treated with Triton X-100 or sonicated. The incorporation was also prohibited by several different treatments of the egg extract resulting in incomplete assembly of the nuclear membrane on the sperm nuclei. On the other hand, there was no inhibition of nuclear incorporation into the sperm nuclei reconstituting in the extracts which had been depleted of WGA-binding pore complex proteins or which contained a specific inhibitor of topoisomerase II (ICRF-193). In these two cases, the nuclear double-layered membrane assembled normally, although in the former case the sperm nuclei lacked lamina and did not initiate DNA replication, and in the latter case the sperm nuclei did not decondense but initiated DNA replication. Thus, it is concluded that DNA polymerase activity is incorporated into the reconstituting nuclei via the membraneous/particulate fraction of the egg extract simultaneously with nuclear double-layered membrane assembly. The lamina assembly and the transport system via the nuclear envelope pore complex are suggested not to participate in DNA polymerase nuclear incorporation.

ICRF-193, an inhibitor of topoisomerase II, demonstrates that DNA replication in sperm nuclei reconstituted in Xenopus egg extracts does not require chromatin decondensation.

A bis(2,6-dioxopiperazine) derivative, ICRF-193, is a specific inhibitor of topoisomerase II without cleavable complex-stabilizing activity. In Xenopus egg extract containing ICRF-193, demembranated sperm head chromatins were inhibited from decondensation. However, nuclear envelope-lamina assembled on the inhibited chromatins. The nuclear envelope-lamina continued to expand even after loss of contact with the chromatin surface. On the other hand, semiconservative DNA replication was initiated as soon as the lamina was assembled onto the surface of condensed chromatin, though the initiation was retarded and its extent was reduced, compared with that in noninhibited chromatins. Thus, it is concluded that topoisomerase II activity is not required for the formation of active DNA replication clusters and the extension of nuclear envelope-lamina on the chromatin, while the nuclear envelope-mediated decondensation of sperm chromatins is dependent on topoisomerase II activity.

Assembly of envelope structure with vesicles associated with Ku-homologous protein in Xenopus egg extract in the absence of chromatin.

To study the process of nuclear envelope assembly at the end of mitosis, we developed a chromatin-free in vitro system for assembly of envelope structures in Xenopus interphase egg extract, and examined the participation of Ku-homologous protein in the assembly. The envelope structure assembled spontaneously in the absence of chromatin or DNA between glass plates under a condition that minimized generation of flow of the extract. Morphological study using an electron microscopy has revealed that the membrane surrounding the envelope structure is a double membrane that contains gaps resembling nuclear pore complex. Their assembly was dependent on ATP and was inhibited by the addition of GTP-gamma-S or N-ethylmaleimide. Depletion of a pre-nuclear vesicle by preincubating the interphase egg extracts with large excess of sperm head chromatins impaired the assembly. The membrane vesicle, which was associated with Ku-homologous protein of Xenopus, participated in the assembly as proven by reaction with monoclonal antibody made specific for Ku p70 protein. However, the assembly process of the envelope structure was inhibited only slightly by the antibody, suggesting that the Ku-homologous protein does not participate in the fusion process of vesicles to form the envelope structure.

A protein homologous to human Ku p70-protein is required for reconstitution of Xenopus sperm pronuclei.

The monoclonal antibody mAbH6, which recognizes one human Ku-protein (p70), cross-reacted with the counterpart protein from Xenopus eggs. The Xenopus antigen purified with a mAbH6-Sepharose column was a complex of 88 kDa and 72 kDa proteins. The role of Ku protein in nuclear structure formation was studied using a cell-free nuclear assembly extract derived from Xenopus interphase eggs. The protein was distributed on the surface of demembranated sperm chromatin and in the membrane vesicle fraction of the nuclear assembly extract. Addition of mAbH6 to the assembly extracts prevented the completion of reconstitution of pronuclei from demembranated sperm chromatins, although partial decondensation mediated by nucleoplasmin was not impeded. As a result, the sperm chromatin remained in partially swollen structures or formed round but small anomalous nuclei. Nuclear membranes were formed on the nuclei in mAbH6-inhibited extract systems, but DNA synthesis was largely decreased, suggesting the incomplete reconstitution of pronuclei. The incorporation of lamin to the nuclei was inhibited by mAbH6. It is suggested that the Xenopus Ku-homologous protein has a role in the formation of the lamin layer after nuclear membrane reconstitution.

Non-homogeneous distribution of beta 1- and beta 2-adrenoceptors in various human tissues.

The distribution of beta 1- and beta 2-adrenoceptor subtypes was determined in membranes obtained from the right atrium, abdominal fat pad, kidney, lung and liver of each of three human subjects. The number of each beta-adrenoceptor subtype was calculated by nonlinear least-squares computer analysis of the competition curve of (-) [125I]iodocyanopindolol (125I-CYP) binding, using beta 1-selective antagonist CGP 20712A and beta 2-selective antagonist ICI 118,551. Competition studies using CGP 20712A indicated that both beta 1- and beta 2-adrenoceptor subtypes coexist in all five tissues, with marked difference in the relative proportion of beta 1- and beta 2-adrenoceptors among these tissues. Most notably, the existence of beta 1-subtypes could be clearly shown in the lung (27%) and liver (18%); according to previous reports, beta 2-subtypes are found almost exclusively in the lung and liver in humans. Regarding the kidney, the present results (beta 2-subtypes: 68%) also differed from previous human data in that beta 1-subtypes predominated. Consistent results were obtained in experiments with ICI 118,551. These results provide a new insight into the mechanisms by which catecholamines regulate a particular beta-adrenergic function in these tissues.

Novel monoclonal antibody mAb G3A5 recognizes 138-kDa glycoprotein localized on the Golgi membrane.

Partially purified Golgi membranes of HeLa cells were used as antigen to produce a novel monoclonal antibody (mAb G3A5). The mAb G3A5 specifically labeled Golgi apparatus of human and monkey cultured cells as ascertained by indirect immunofluorescence but did not stain those of bovine or mouse cells. Treatment with nocodazole and brefeldin A (BFA) induced fragmentation and redistribution of the staining. Western immunoblot analysis showed that mAb G3A5 was directed against a single polypeptide with an apparent molecular mass of 138-kDa (p138 antigen). The p138 antigen is an integral membrane protein of the Golgi apparatus, as assessed by several assays: protease protection, salt wash and flotation in sucrose density gradient centrifugation. The p138 antigen was purified using immunoaffinity chromatography. The apparent molecular mass of the p138 antigen decreased by 2 to 4 kDa after treatment with the peptide: N-glycosidase F, while digestion with ENDO F or Neuraminidase did not have this effect. Thus, p138 antigen is a glycoprotein containing asparagine-linked carbohydrates.

Immunolocalization of Ku-proteins (p80/p70): localization of p70 to nucleoli and periphery of both interphase nuclei and metaphase chromosomes.

Distribution on both nuclei and metaphase chromosomes of Ku-proteins, recognized by autoantibodies from a patient with systemic lupus erythematosus, has been studied using a specific monoclonal antibody (mAbH6) that recognizes p70, one Ku-protein. Observation with either a conventional fluorescent microscope or a confocal laser scanning microscope revealed mAbH6-stained p70 antigen localized on both nuclear periphery and nucleoli of human interphase cells. The specific staining of nucleoli with mAbH6 has been confirmed using isolated nucleoli from rat liver in which the staining was seen as fine granules surrounding nucleolar DNA. During mitosis p70 antigen moved away from association with the nuclear envelope region to localization on the periphery of condensed chromosomes with no apparent staining of chromosome interior. The p70 antigen was copurified with DNA fragments by immunoaffinity column chromatography using mAbH6. The mAbH6 staining of both nuclear periphery and nucleoli was lost upon digestion with DNase I at low concentrations. These results suggest that p70 antigen is connected with these nuclear structures through DNA.

Structure of DNA polymerase alpha-primase complexes from mammalian cells analyzed by using monoclonal antibodies.

The molecular masses of two of the four DNA polymerase alpha-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase alpha-primase complexes. Neither of these antibodies reacts with DNA polymerase alpha-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecular masses of the two main peptides of DNA polymerase alpha-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.

Multivariable analysis of serum 3,5,3'-L-triiodothyronine concentration in patients of diabetes mellitus by blood glucose level and body weight.

Serum thyroid hormone concentrations, 1-thyroxine (T4), free T4 and 3,5,3'-l-triiodothyronine (T3) were measured in 213 patients of diabetes and analyzed their correlation with metabolic parameters, hyperglycemia and body weight. Haemoglobin A1 (HbA1) was used as an index of hyperglycemia. Body weight was expressed by relative body weight (body weight/standard weight). Among the thyroid hormones, only T3 had significant correlation with HbA1 and body weight (r = -0.476, P less than 0.01 and r = 0.369, P less than 0.01, respectively). Multivariable analysis of serum T3 by HbA1 and relative body weight gave the following regression equation. Serum T3 (ng/dl) = 108 + 0.362 x relative body weight (%) - 3.88 x HbA 1 (%). Though relative body weight had inverse correlation with HbA1, the contribution of the two metabolic parameters to the serum T3 was independent from each other. Our results confirm the previous reports that low T3 in diabetes correlates with severity of hyperglycemia and we report for the first time that serum T3 of diabetic patients has positive correlation with body weight, probably due to still available carbohydrate in spite of disturbances in the metabolism.

Intracellular localisation of a subunit of human DNA polymerase alpha affecting primase activity recognised by monoclonal antibody (HDR-854-E4) and its application to distinction between proliferative and non-proliferative lesions.

We have successfully established one murine hybridoma that secretes a monoclonal antibody specific for the 77,000 subunit of human DNA polymerase alpha. The results of immunochemical studies, using HDR-854-E4 monoclonal antibody (MAb) and immunoperoxidase detection methods, demonstrate intranuclear and intracytoplasmic localisation of the subunit in all the human culture cell lines tested. The immunoperoxidase reaction product exhibits a diffuse pattern of distribution within the cytoplasm and nucleoplasm, but nucleoli are clearly negative. In cultured cell lines, HeLa and KATO III, more than 95% of the cells are positive, suggesting that the subunit antigens persist throughout the mitotic cycle. No subunit antigen was recognised in resting mononuclear cells (MNC). Immuno-electron microscopic examination of HeLa cells confirms and extends these observations. We have further examined the expression level of the subunit antigen in various normal and cancerous tissues. Strong reaction was observed in proliferating normal and cancer cells such as cancer cells from the gastrointestinal (GI) tract, thyroid, malignant lymphoma, breast, cells in the germinal centres of lymph nodes, epithelial cells in the GI tract and nephrogenic zones in fetal kidney. Finally, we utilised this antibody as a diagnostic tool in biopsies of the thyroid and GI tract. Thyroid cancer was stained positively with this antibody, while follicular adenoma was not. Gastric cancer was stained strongly and adenomatous polyp and hyperplastic polyp were stained moderately. This antibody is not only specific and powerful for application of a novel approach to the complex biochemical mechanisms of mammalian DNA replication, but also useful for distinction between proliferative and non-proliferative lesions.