Albino inflorescence proliferation of Dendrocalamus latiflorus

Summary Inflorescence proliferation is a plant tissue culture technique that, can be used to obtain in vitro inflorescences year-round without the intervening development of vegetative organs. In this study, we used albino mutant inflorescences of Dendrocalamus latiflorus as the original explant material to investigate, the effect of plant growth regulators on long-term inflorescence proliferation. The albino inflorescences proliferated on solidified Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ), and the optimal concentration for successful long-term inflorescence proliferation was 0.45 μM TDZ. A combination of α-naphthaleneacetic acid (NAA) with 0.45 μM TDZ inhibited the inflorescence proliferation. Inflorescences cultured on a TDZ-free medium supplemented with 26.82 μM NAA rooted in 21 d, vegetative shoots formed by 42 d and, in one case, flowering occurred after 63 d. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D, 4.52 μM) and pieloram (4.14 μM) induced shoot formation. The protocol described can be used to produce large numbers of mutant inflorescences within a relatively short period of time.     

N.m.r. and computer-simulated conformational analyses of a nonapeptide found in a human salivary proline-rich glycoprotein

The amino acid sequence G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9) occurs twice in the proline-rich glycoprotein (PRG) found in human parotid saliva. As part of our efforts to elucidate the structure-function relationships of PRG, this nonapeptide sequence (PRG9) was synthesized for the purpose of conformational analyses by high-resolution proton n.m.r. spectroscopy and computer-modeling. The empirical n.m.r. spectrum differed from the simulated spectrum in that the overall chemical shift locations were displaced from their random coil positions and the five proline residues had non-degenerate C alpha H alpha protons. Other n.m.r. data indicated that no intramolecular hydrogen-bonding was present in the PRG. In conjunction with X-ray crystallographic data on a triproline-containing model compound (Kartha, g., Ashida, T. & Kakudo, M. (1974) Acta Cryst. B30, 1861-1866), four energy-minimized PRG9 structures were obtained. Two of the structures were energetically unfavorable, while the other two conformations were reasonable. The two most likely structures gave all prolines an S-type ring pucker, the P(2)-P(3)-P(4) sequence as a poly-L-proline II helix, the H(5) phi = -90.3 degrees, P(6) and P(9) with trans peptide bond orientation, G(7) in an extended state, and the K(8) phi = -93.2 degrees or -146.8 degrees for structures #1 and #2, respectively.     

Mitogenomic analysis of Montipora cactus and Anacropora matthai (cnidaria; scleractinia; acroporidae) indicates an unequal rate of mitochondrial evolution among Acroporidae corals

The complete nucleotide sequence of the mitochondrial (mt) genome was determined for specimens of the coral species Montipora cactus (Bernard 1897) and Anacropora matthai (Pillai 1973), representing two morphologically distinct genera of the family Acroporidae. These sequences were compared with the published mt genome sequence for the confamilial species, Acropora tenuis (Dana 1846). The size of the mt genome was 17,887 bp and 17,888 bp for M. cactus and A. matthai. Gene content and organization was found to be very similar among the three Acroporidae mt genomes with a group I intron occurring in the NADH dehyrogenase 5 (nad5) gene. The intergenic regions were also similar in length among the three corals. The control region located between the small ribosomal RNA (ms) and the cytochrome oxidase 3 (cox3) gene was significantly smaller in M. cactus and A. matthai (both 627 bp) than in A. tenuis (1086 bp). Only one set of repeated sequences was identified at the 3′-end of the control regions in M. cactus and A. matthai. A lack of the abundant repetitive elements which have been reported for A. tenuis, accounts for the relatively short control regions in M. cactus and A. matthai. Pairwise distances and relative rate analyses of 13 protein coding genes, the group I intron and the largest intergenic region, igr3, revealed significant differences in the rate of molecular evolution of the mt genome among the three species, with an extremely slow rate being seen between Montipora and Anacropora. It is concluded that rapid mt genome evolution is taking place in genus Acropora relative to the confamilial genera Montipora and Anacropora although all are within the relatively slow range thought to be typical of Anthozoa.     

Ectopic Expression of the Tetratricopeptide Repeat Domain of SPINDLY Causes Defects in Gibberellin Response

The SPINDLY (SPY) protein of Arabidopsis is a negative regulator of gibberellin (GA) response. The SPY protein has 10 copies of the tetratricopeptide repeat (TPR) at the N terminus. TPR motifs function as protein-protein interaction domains. Several spy alleles are affected only in the TPR region suggesting that protein-protein interactions mediated by this domain are important for proper GA signaling. We have used a reverse genetics approach to further investigate the role of the TPR domain. The TPR domain of SPY was overexpressed in wild-type, gai, and spy plants. Expression of the TPR domain alone is not sufficient to rescue spy mutants. Expression of the TPR domain in a wild-type background produces phenotypes similar to those caused by loss-of-function spy mutants including resistance to GA biosynthesis inhibitors, short hypocotyl length, and early flowering. The dwarfing of the floral shoot internodes caused by the gai mutation was suppressed by expression of the TRP domain. Expression of the TPR domain had no effect on the abundance of endogenous SPY mRNA. The TPR domain was found to interact with SPY both in vitro and in yeast two-hybrid assays. These data indicate that the TPR domain of SPY can participate in protein-protein interactions and that these interactions are important for the proper functioning of SPY.     

Carrier detection and prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction

Summary Alpha-thalassemia of Southeast Asian deletion (-- SEA/) is very common in Southeast Asia. Homozygosity of this genotype is the major cause of Hb Bart's hydrops fetalis in Taiwan. With polymerase chain reaction using three oligonucleotide primers bridging the common deletion breakpoint, a DNA fragment of 194 basepairs (bp) was amplified in chromosomes with the-- SEA determinant and a DNA fragment of 287 bp was amplified in chromosomes without this deletion. In our pilot study including 8 normal subjects, 20 obligate carriers, and 11 homozygotes of the deletion, all the genotypes were determined and then confirmed by Southern blotting and DNA hybridization with globin gene probe. For prenatal diagnosis, 55 at-risk pregnancies were collected. Chorionic villus sampling was done in 51 cases and early amniocentesis was done in 4 cases. Fourteen cases (25.5%) were diagnosed as normal, 25 (45.5%) as heterozygotes, and 16 (29%) as homozygotes of -- SEA. All of the diagnoses were also confirmed as aforementioned. With polymerase chain reaction, the determination of the -- SEA deletion is straightforward and is much quicker and easier than with conventional Southern blotting and DNA hybridization. In areas with a high prevalence of -- SEA deletion, this method provides a rapid tool for carrier detection and prenatal diagnosis.     

The human cationic amino acid transporter (ATRC1): physical and genetic mapping to 13q12-q14.

The product of the mouse Rec-1 locus is an integral membrane protein that determines susceptibility to infection by murine ecotropic retroviruses. Recently it has been determined that its role in normal cell metabolism is transport of the cationic amino acids, arginine, lysine, and ornithine across the plasma membrane. Southern blot analysis of genomic DNA from a panel of 48 mouse-human somatic cell hybrids assigned the human version of this gene, ATRC1, to chromosome 13. Chromosomal in situ hybridization localized the gene to 13q12-q14. A restriction fragment length polymorphism (RFLP) was detected with TaqI. There were two alleles with frequencies of 0.29 and 0.71. Pairwise linkage analysis established linkage between ATRC1 and ATP1AL1, D13S1, D13S6, D13S10, D13S11, D13S21, D13S22, D13S33, D13S36, and D13S37. Multilocus linkage analysis of five of the loci indicated that the most likely order of loci in this region was D13S11-ATP1AL1-ATRC1-D13S6-D13S33.     

Characterization of alpha-actinin from Acanthamoeba

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.     

The fetal rat binding protein for insulin-like growth factors is expressed in the choroid plexus and cerebrospinal fluid of adult rats

The biological effects of the insulin-like growth factors, IGF-I and IGF-II, on their receptors are modulated by IGF-binding proteins. Recently, we isolated a cDNA clone for one member of the family of IGF-binding proteins, BP-3A, a 30 kilodalton (kDa) protein synthesized by the BRL-3A rat liver cell line. BP-3A is related to but distinct from two other cloned IGF-binding proteins, the human amniotic fluid binding protein and the glycosylated binding subunit of the 150 kDa IGF-binding protein complex in serum. It is expressed in multiple nonneural tissues and in serum in the fetal rat and decreases after birth, similar to the developmental pattern of IGF-II expression. IGF-I, IGF-II, and their receptors are expressed in brain. The present study examines the expression of BP-3A in the rat central nervous system. By Northern blot analysis, BP-3A mRNA is present at high levels in brain stem, cerebral cortex, and hypothalamus from 21-day gestation rats and, like IGF-II mRNA, persists in adult rat brain. The site of BP-3A mRNA synthesis was localized by in situ hybridization to coronal sections of adult rat brain using 35S-labeled oligonucleotides, 48 bases in length, complementary and anticomplementary to the coding region of BP-3A. Specific hybridization of the BP-3A probe was observed exclusively to the choroid plexus extending from the level of the medial preoptic nucleus to the arcuate nucleus of the hypothalamus, similar to the previously reported preferential localization of IGF-II mRNA to the choroid plexus. Synthesis of BP-3A mRNA by choroid plexus suggested that BP-3A might be secreted into the cerebrospinal fluid. A 30 kDa IGF-binding protein was demonstrated in rat cerebrospinal fluid that is recognized by antibodies to BP-3A and, like purified BP-3A, has equal affinity for IGF-I and IGF-II. By analogy with other transport proteins synthesized by the choroid plexus, BP-3A may facilitate the secretion of IGF-II to the cerebrospinal fluid and modulate its biological actions at distant sites within the brain.     

Immunologic identification of the aromatase enzyme system in human endometrium

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.