Ancient mitochondrial DNA reveals the origin of Sus scrofa from Rebun Island, Japan.

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north-northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences.

Neural circuits and functional organization of the striatum.

The basal ganglia and motor thalamic nuclei are functionally and anatomically divided into the sensorimotor, supplementary motor, premotor, associative and limbic territories. There exist both primary segregated basal ganglia-thalamocortical loops and convergence of functionally related information from different cortical areas onto these cortical basal ganglia-thalamocortical loops. The basal ganglia-thalamocortical loop arising from the sensorimotor area, supplementary motor area (SMA), premotor area and cingulate motor area provides distinct segregated subloops through the functionally distinct striatal, pallidal and thalamic regions with partial overlap. The subthalamic nucleus (STN) is also topographically organized. The ventrolateral part of the caudal 2/3 levels of the medial pallidal segment (GPi) projects to the primary motor area via the oral part of the ventral lateral thalamic nucleus (VLo) (Voa, Vop by Hassler's nomenclature). The thalamic relay nuclei of the GPi projection to SMA are identified in the transitional zone of the VApc (parvicellular part of the anterior ventral nucleus)-VLo and in the rostromedial part of the VLo. The thalamic nuclei relaying the cingulate subloop are not yet clearly defined. The supplementary motor subloop appears to be divided into the pre-SMA and SMA proper subloops. The premotor area is also divided into the dorsal premotor area subloop and the ventral premotor area subloop. It is suggested that the limbic loop consists of a number of subloops in the monkey as indicated by Haber et al. and in rats. We review here the microcircuitry of the striatum, as well as the convergence and integration between the functionally segregated loops. Finally, we discuss the functional implications of striatal connections.

Proline- and alanine-rich Ste20-related kinase associates with F-actin and translocates from the cytosol to cytoskeleton upon cellular stresses.

Proline- and alanine-rich Ste20-related kinase (PASK) is a Ste20-related protein kinase isolated from rat brain. Cell fractionation studies showed that PASK was present both in the cytosol and in Triton X-100-insoluble cytoskeletal fraction in rat tissues. In brain, PASK associated with protein complexes that contained actin and tubulin, confirming the association of PASK with the cytoskeleton in vivo. Glutathione S-transferase-PASK fusion protein cosedimented with F-actin, indicating that PASK binds to F-actin. In contrast to rat tissues, PASK was detected only in the Triton X-100-soluble cytosolic fraction in cultured PC12 and NIH 3T3 cells. Cytosolic PASK translocated to the cytoskeleton when these cells were stimulated with severe cellular stresses such as hypertonic sodium chloride, hydrogen peroxide, and heat shock at 45 degrees C. Our results suggest that PASK may be involved in the regulation of the cytoskeleton in response to cellular stresses such as hyperosmotic shock.

Molecular cloning and characterization of a novel Ste20-related protein kinase enriched in neurons and transporting epithelia.

A novel cDNA encoding a protein kinase (termed PASK) was isolated from rat brain. The PASK catalytic domain was most similar to Ste20-related protein kinases, showing 45.5 and 39.2% amino acid identity with human SOK1 and yeast Sps1, respectively. The amino-terminal noncatalytic domain of 71 amino acids was rich in alanine and proline and contained several proline-alanine repeats. PASK was widely expressed in rat tissues but negligible in liver and skeletal muscle. Immunohistochemical analysis revealed that PASK was localized to a distinct set of cells including neurons, adrenal glomerulosa cells, and transporting epithelia such as epithelial cells of brain choroid plexus, distal tubule and collecting duct of kidney, duct of salivary gland, and parietal cells of stomach. Subcellular fractionation showed that PASK was present in both the cytosol and the Triton X-100-insoluble cytoskeletal fraction in brain.

Differential regulation of prolactin receptor mRNA expression in rat liver and kidney by testosterone and oestradiol.

Prolactin (PRL) exerts a wide variety of physiological effects on mammalian tissues through its receptor (PRL-R) on the target cells. PRL-R in rat tissue consists of two isoforms, the long and the short form, and the regulatory mechanisms of their mRNA expression in tissues are complex and diverse. The present study reports the differential regulation of PRL-R mRNA expression in rat liver and kidney by testosterone and oestradiol. Using Northern blot analysis, short form PRL-R mRNA was clearly detected in female rat liver and male rat kidney, and long form PRL-R mRNA was faintly observed only in female rat liver. However, the reverse transcription-polymerase chain reaction method enabled efficient analysis of mRNA levels in short and long forms of PRL-R in the liver and kidney of both male and female rats. The mRNA levels for the long and short forms of PRL-R were depressed in the liver of male rats but not in that from female rats during sexual maturation. Castration of male rats resulted in the induction of the mRNAs for these two forms of PRL-R in the liver. Testosterone, but not oestradiol, completely blocked the induction by castration of liver PRL-R gene expression. In kidney, in contrast, mRNA levels for both forms of PRL-R were depressed in female rats but not in male rats after sexual maturation. Administration of oestradiol, but not of testosterone, caused marked repression of short form PRL-R mRNA, particularly in the kidney of male rats. The levels of long form PRL-R mRNA in the kidney was less affected by the administration of oestradiol. These results have suggested that the expression of PRL-R mRNAs in rat liver and kidney is differentially regulated by testosterone and oestrogen.

Syntheses of recombinant yellowtail and flounder growth hormones in Escherichia coli.

For syntheses of recombinant yellowtail and flounder growth hormones (r-yGH and r-fGH) in E. coli, expression plasmids were constructed. The expression level of r-yGH and r-fGH in the host cells were very high, reaching 15 and 8% of the total protein, respectively. These product proteins were accumulated in inclusion bodies in the cells. The recombinant hormones were isolated from the pellets ina glutathione reduction/oxidation buffer. The refolded hormones were further purified by DEAE-Toyopearl 650M chromatography to homogeneity. The purified r-yGH and r-fGH were composed of 188 and 174 amino acid residues, respectively, having amino-terminal sequences starting with methionine. The recombinant hormones had potent growth-promoting activities on juvenile rainbow trout Salmo gairdneri in a dose-dependent manner.

A novel cDNA clone encoding a prolactin-like protein that lacks the two C-terminal cysteine residues isolated from bovine placenta.

A new prolactin-like cDNA clone, bPLP-IV, was isolated from a bovine placental cDNA library and the complete nucleotide sequence was determined. The bPLP-IV encodes a protein consisting of 237 amino acids, which is related to, but different from seven other known bovine prolactin-like proteins including two placental lactogens. The predicted amino acid sequence of the bPLP-IV shows over 52% identity to other known members of bovine prolactin-like proteins, 48% to bovine prolactin, 40% to both two bovine placental lactogens and only 22% to bovine growth hormone. The bPLP-IV protein has a unique feature in its primary structure, lacking the two C-terminal cysteine residues which are completely conserved in all other known members of prolactin-growth hormone-placental lactogen gene family. The expression of bPLP-IV in developing bovine placenta was apparently stage-specific, being maximal in the full-term placenta.

Human poly(ADP-ribose) polymerase gene. Cloning of the promoter region.

The promoter region of the poly(ADP-ribose) polymerase gene has been isolated using a Sau3AI genomic library derived from human leukocyte. It lacks typical transcriptional regulatory elements such as TATA and CAAT boxes, but it contains two potential Sp1 binding sites and three putative AP-2 binding elements. The region up to nucleotide position-99 in relation to the predominant transcriptional initiation site exhibits promoter activity as judged by chloramphenicol acetyltransferase assay and the activity is enhanced both by cAMP and by phorbol ester. Northern blot and Western blot analyses have revealed that expression of the polymerase gene is also stimulated by both of these compounds in cultured HeLa cells. Southern blot hybridization of genomic DNA separately digested with various endonucleases gives a discrete single band in each case when the 5'-untranslated region of the polymerase cDNA is used as a probe. These results indicate that poly(ADP-ribose) polymerase is encoded by a unique gene whose expression is regulable by cAMP and by phorbol ester.

Alternative usage of different poly(A) addition signals for two major species of mRNA encoding human aromatase P-450.

Two cDNA clones for human placental aromatase P-450 (P-450AROM) have been isolated and sequenced. The insert of one clone (2894 bp) contains an open reading frame encoding a protein consisting of 503 amino acid residues together with a 49 bp 5'-untranslated stretch and a 1336 bp 3'-noncoding region to which a poly(A) tract is attached. Three potential poly(A) addition signals are detected in this 3'-noncoding region. The other clone contains a shorter cDNA insert, the nucleotide sequence of which overlaps with most of the sequence of the longer cDNA insert (nucleotides 36-2355) except for one nucleotide substitution. The 3'-noncoding region of this shorter cDNA is only 846 bp in length but a poly(A) tract is also attached to its 3'-terminus. Northern blot analysis of human placental RNA reveals the presence of two major mRNA species of 3.4 and 2.9 kb when probes excised from the overlapping region of these two cDNAs are employed. The 2.9 kb mRNA is not detected, however, when a fragment of the non-overlapping region of the longer cDNA is used as a probe. It is therefore concluded that the two major species of P-450AROM mRNA are formed as a consequence of alternative processing of precursor mRNA(s).

Primary structure of human poly(ADP-ribose) synthetase as deduced from cDNA sequence.

Human poly(ADP-ribose) synthetase consists of three proteolytically separable domains, the first for binding of DNA, the second for automodification, and the third for binding of the substrate, NAD (Ushiro, H., Yokoyama, Y., and Shizuta, Y. (1987) J. Biol. Chem. 262, 2352-2357). We have isolated and sequenced cDNA clones for the enzyme using synthesized oligodeoxyribonucleotide probes based on the partial amino acid sequence of the protein. The open reading frame determined encodes a protein of 1,013 amino acid residues with a molecular weight of 113,203. The deduced amino acid sequence is consistent with the partial amino acid sequences of tryptic or alpha-chymotryptic peptides and the total amino acid composition of the purified enzyme. The native enzyme is relatively hydrophilic as judged from the hydrophilicity profile of the total amino acid sequence. The net charge of the NAD binding domain is neutral but the DNA binding domain and the automodification domain are considerably rich in lysine residue and quite basic. The DNA binding domain involves a homologous repeat in the sequence and exhibits a sequence homology with localized regions of transforming proteins such as c-fos and v-fos. Furthermore, this domain contains a unique sequence element which resembles the essential peptide sequences for nuclear location of SV40 and polyoma virus large T antigens. These facts suggest the possibility that the physiological function of poly(ADP-ribose) synthetase lies in its ability to bind to DNA and to control transformation of living eukaryotic cells like the cases of those oncogene products.

Purification and characterization of poly (ADP-ribose) synthetase from human placenta.

Poly(ADP-ribose) synthetase has been purified 2,000-fold to apparent homogeneity from human placenta. The purification procedure involves affinity chromatography with 3-aminobenzamide as the ligand. The purified enzyme absolutely requires DNA for the catalytic activity and catalyzes poly(ADP-ribosyl)ation of the synthetase itself (automodification) and histone H1. Mg2+ enhances both the automodification and poly(ADP-ribosyl)ation of histone H1. The enzyme is a monomeric protein with a pI of 10.0 and an apparent molecular weight of 116,000. The sedimentation coefficient and Strokes radius are 4.6 S and 5.9 nm, respectively. The frictional ratio is 1.82. Amino acid analysis and limited proteolysis with papain and alpha-chymotrypsin indicate that the human placental enzyme is very similar to the enzyme from calf thymus, although some differences are noted. Mouse antibody raised against the placental enzyme completely inhibits the activity of enzymes from human placenta and HeLa cells and cross-reacts with the enzymes from calf thymus and mouse testis. Immunoperoxidase staining with this antibody demonstrates the intranuclear localization of the enzyme in human leukemia cells. All these results indicate that molecular properties as well as antigenic determinants of poly(ADP-ribose) synthetase are highly conserved in various animal cells.

Effects of alpha-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes.

The effect of alpha-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of alpha-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. beta-, gamma-, and delta-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 +/- 0.12 microM) of the interaction of alpha-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of alpha-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 microM ascorbic acid and 10 microM Fe2+. Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by alpha-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 +/- 0.16 to 18.5 +/- 0.51 microseconds after addition of 25 microM alpha-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by alpha-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl+. Based on these results, the effect of alpha-tocopherol on the lipid fluidity of the membranes is discussed.

Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP-ribose) synthetase.

Calf thymus poly(ADP-ribose) synthetase (Mr = 120,000) is cleaved with papain into two fragments of M(r) = 74,000 and 46,000 and also split with chymotrypsin into two fragments of M(r) = 66,000 and 54,000. Each fragment purified to homogeneity is enzymatically inactive, but combined incubation of the 74,000 and 46,000 fragments in the presence of DNA restored 20% of the enzyme activity. In contrast, combined incubation of the 66,000 and 54,000 fragments does not restore any enzyme activity. In the former incubation, autopoly(ADP-ribosyl)ation reaction occurs exclusively on the 74,000 fragment. When each fragment is incubated with [adenine-U-14C]NAD in the presence of DNA and a catalytic amount of the native enzyme, poly(ADP-ribosyl)action occurs in the overlapped portion (22,000) of the 66,000 fragment and the 74,000 fragment. Nevertheless, the purified 22,000 fragment is a poor acceptor for poly(ADP-ribosyl)ation. The degree of poly(ADP-ribosyl)ation of the proteolytic fragments is significantly reduced by increasing NaCl concentration, probably due to the lack of the interaction between the enzyme fragments and DNA. These results, taken together, indicate that DNA is indispensable for the reconstitution of the catalytic activity as well as the poly(ADP-ribosyl)ation of the fragmented enzyme.

The domain structure and the function of poly(ADP-ribose) synthetase.

Poly(ADP-ribose) synthetase is a chromatin-bound enzyme which synthesizes a protein-bound homopolymer of ADP-ribose utilizing NAD as a substrate. The characteristic nature of this enzyme is that it requires DNA for catalytic activity. The enzyme is rich in malignant tumor cells as well as in normal tissues where cell proliferation is very rapid. The enzyme has been purified to homogeneity from calf thymus, mouse testis and human placenta. The amino acid composition of these enzymes is very similar and a monoclonal antibody as well as antisera against the calf enzyme cross-reacts with mouse, chicken and human enzymes, suggesting that the antigenic structures of poly(ADP-ribose) synthetase are highly conserved in various animal cells. The native enzyme (Mr = 120K) is cleaved by limited proteolytic digestion into three different domains (Mr = 46K, 22K, 54K), the first containing the site for DNA binding, the second containing the site for automodification and the third containing the site for NAD binding. The DNA binding domain (Mr = 46K), like the native enzyme, has the ability to preferentially suppress nick induced random transcription initiation in a HeLa cell lysate, resulting in the production of run-off RNA initiated from the correct late promoter site on truncated DNA of adenovirus 2. The native enzyme poly(ADP-ribosyl)ates RNA polymerase and some other nuclear enzymes. These results, taken together, indicate that poly(ADP-ribose) synthetase plays a critical role in regulating gene expression in various eukaryotic cells.

A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles.

A method is presented for the preparation of a "native" epidermal growth factor (EGF) receptor-kinase complex of molecular weight 170,000 from A-431 cells. Although this receptor complex is capable of binding EGF, noncovalently, in quantities similar to the previously isolated 150,000 complex (Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem. 255, 4834-4842), the 170,000 preparation has 5 to 10 times the intrinsic kinase activity (autophosphorylation). However, the 170,000 kinase activity toward other proteins is lower than that of the 150,000 preparation. Both the 170,000 and 150,000 kinase activities are enhanced by EGF. The 170,000 and 150,000 proteins are also capable of forming covalent linkages to 125I-EGF, and each is precipitated by antisera directed against the 170,000 protein. We suggest the 150,000 protein is a proteolytic degradation product of the 170,000 protein. The EGF-enhanced kinase activity of the 170,000 preparation remains associated with the 125I-EGF-binding activity following EGF affinity chromatography, electrophoresis in nondenaturing gels, or immunoprecipitation with antisera directed against the sodium dodecyl sulfate (SDS) gel-purified 170,000 protein. These results indicate that the receptor, kinase, and substrate domains are linked, possibly covalently.

Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cell membranes.

Epidermal growth factor (EGF)-enhanced protein kinase activity in plasma membrane preparations of A-431 human epidermoid carcinoma cells was shown to involve the phosphorylation of tyrosine residues. Phosphotyrosine was detected in both endogenous membrane proteins and in histone when added as an exogenous protein substrate. The major phosphorylated amino acid in partial acid hydrolysates of 32P-labeled A-431 membranes was identified as phosphotyrosine on the basis of its identical behavior to authentic phosphotyrosine on paper electrophoresis and thin layer chromatography; its 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivative was indistinguishable from that of the authentic compound. Only traces, if any, of phosphoserine or phosphothreonine were detected. [32P]Phosphotyrosine was also detected in pronase digests of 32P-labeled membrane proteins. The EGF receptor . protein kinase complex, which was solubilized with Triton X-100 and purified by EGF affinity chromatography, was shown to phosphorylate tyrosine residues of the isolated membrane protein.

Isolation and reconstitution of two electron transfer components of tryptophan side chain oxidase.

The isolation and reconstitution of two electron transfer components of tryptophan side chain oxidase from Pseudomonas (ATCC 29574) are described. The dehydrogenase component abstracts electrons from the substrate and transfers them to oxidation-reduction dyes such as potassium ferricyanide and 2,6-dichlorophenolindophenol but not to molecular oxygen. It is composed of a single polypeptide chain with a molecular weight of 72,000 and exhibits the absorption spectrum of a reduced b-type cytochrome with maxima at 563, 532, 433, 323, and 278 nm. The oxidase component transfers electrons, derived from the former component, to oxygen, and has a molecular weight of 48,000. The absorption spectrum exhibits broad peaks at 680, 438, and 358 nm, and a peak at 280 nm. On sucrose gradient centrifugation and polyacrylamide gel electrophoresis, these two components are shown to form a molecular complex, which has the reconstituted oxidase activity. The turnover number of the reconstituted enzyme is comparable to that of the native enzyme.