P1 nuclease defines a subpopulation of active SV40 chromatin--a new nuclease hypersensitivity assay.

Under exhaustive digestion conditions P1 nuclease was found to cleave a subpopulation of intracellular SV40 chromatin only once. The major P1 cleavage site in SV40 DNA was mapped at the origin of DNA replication, and the two minor sites at the SV40 enhancers. The P1-sensitive SV40 chromatin subpopulation was found to have higher superhelical density than the bulk of the intracellular SV40 chromatin. Furthermore, pulse labeled SV40 DNA which had higher superhelical density than that of the steady state viral DNA (S.S.Chen and M.T.Hsu, J.Virol 51:14-19, 1984) was also found to be preferentially cleaved by P1 nuclease. These results are consistent with a supercoil-dependent alteration of chromatin conformation near the regulatory region of the viral genome that can be recognized by P1 nuclease. Since P1 nuclease cleaves the subpopulation of SV40 chromatin only once without further degradation, this nuclease can be used as a general tool to define viral or cellular chromatin fraction with altered chromatin conformation and to map nuclease hypersensitive sites. Preliminary studies indicate that P1 makes limited double stranded cleavages in cellular chromatin to generate large DNA fragments.     

Hypoxanthine guanine phosphoribosyltransferase deficiency: nucleotide substitution causing Lesch-Nyhan syndrome identified for the first time among Japanese

Summary A previously undescribed nucleotide substitution at codon 51 (CGA to TGA) has been identified using the polymerase chain reaction technique in hypoxanthine guanine phosphoribosyltransferase (HPRT) cDNA; this is the first molecular evidence for a point mutation in a Japanese patient with Lesch-Nyhan syndrome. The present mutation is the 19th nucleotide substitution identified as a germ-line mutation at this locus and the second mutation generating a stop codon. The position of the nucelotide substitution is exactly the same as a previously described mutation HPRT_Toronto, indicating for the first time that nucleotide substitutions at the same position in the sequence of HPRT can generate different mutant alleles, one causing a partial deficiency and the other a complete deficiency. Although the type of nucleotide substitution is different between the two cases, a single base position has twice become the target of a mutation. However, the calculation of the probability of finding substitution mutations at the same base position in the coding region of hprt indicates that there is no evidence for the presence of a hot spot for substitution mutations in the human hprt germ line.     

Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.     

Plant regeneration in vitro of South Pacific taro (Colocasia esculenta var. esculenta cv. Akalomamale,Aracea)

Summary Axillary bud expiants from South Pacific (Solomon Islands) taro, Colocasia esculenta var. esculenta cv. Akalomamale (Araceae) cultured on a modified Murashige-Skoog medium containing 1 mg NAA 1^–1 and TE formed callus and produced multiple plantlets. Explants died if NAA was present at levels lower than 0.1 mg 1^–1. BA was not required and may have been inhibitory. Plantlets developed faster and became larger following transfer to a hormone-free medium two weeks after the start of culture. Fully grown plants were established in a potting mix and are growing well in a greenhouse.Abbreviations BA benzyladenine - BM basal medium - Ca Colocasia esculenta var. antiquorum - Ce Colocasia esculenta var. esculenta - Ck cytokinin(s) - CW coconut water - HSMSM half strength Murashige Skoog macroelements - HSMS half strength Murashige and Skoog medium - IM initial medium(ia) - MS Murashige and Skoog medium - NAA naphthaleneacetic acid - SM second medium - TE taro corm extract - UCI University of California, Irvine     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

A partial map of the barley genome incorporating restriction fragment length polymorphism, polymerase chain reaction, isozyme, and morphological marker loci

Nine low copy number genomic DNA clones, a ribosomal sequence, and seven cDNA clones were found to identify polymorphisms in cultivated barley (Hordeum vulgare L.). An F2 population consisting of 100 plants was produced from a cross between a high-yielding two-rowed feed barley cultivar and a genetic marker stock homozygous for nine recessive and one dominant morphological marker genes. Through the use of these 10 well-distributed marker genes, five previously mapped isozyme loci, and two storage-protein loci, the approximate recombinational location for each of 17 restriction fragment length polymorphism loci was estimated. One clone, pMSU21, identified variation that appeared to be the result of a small insertion-deletion event that differentiated two-rowed and six-rowed genotypes. This difference was characterized, and one allele was sequenced. Oligonucleotide primers that flanked the insertion-deletion event were synthesized, and DNA samples from the F2 population were subjected to polymerase chain reaction sequence amplification. The variation identified by this technique was determined to be allelic to the variation identified using pMSU21 in Southern blot analysis. Maps of previously undescribed informative clones are included.     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

Electron-microscopic studies on the threshold value of calcium concentration for the release of storage granules and the acceleration of their degradation in the rat parathyroid gland

Summary To determine both a threshold value of calcium concentration (CC) for the release of storage granules and that for the acceleration of degradation of these granules, the rat parathyroid glands were perfused in situ with HEPES-Ringer solutions containing different concentration of Ca²⁺ for 10 min. With perfusates containing 0.83–1.21 mM Ca²⁺ (equivalent to 8–11 mg/dl serum calcium), the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged. With perfusates containing 0.83 mM Ca²⁺ (7.5 mg/dl) or less, however, both NSG-I and NSG-II decreased remarkably and the former was larger than the latter. On the contrary, with perfusates containing 1.27 mM Ca²⁺ (11.5 mg/dl) or more, NSG-II increased and the ratio of NSG-I to NSG-II was changed reversely. We concluded that a thereshold value of CC required for the release of storage granules may be present between 0.88 and 0.83 mM Ca²⁺ (8 and 7.5 mg/dl) and that a threshold value of CC for accelerating the transformation of type-I granules into type-II, the degradation of storage granules, may be situated at about 1.27 mM Ca²⁺ (11.5 mg/dl). Additionally, it was suggested that both prosecretory and storage granules are not only formed at the innermost Golgi cisterna but also at the trans-Golgi network.     

Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.     

Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRTAnn Arbor)

Summary HPRT_{Ann Arbor} is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC 2.4.2.8), which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased K_m for both substrates, a normal V_max, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric focusing conditions. We have cloned a full-length cDNA for HPRT_{Ann Arbor} and determined its complete nucleotide sequence. A single nucleotide change (TG) at nucleotide position 396 has been identified. This transversion predicts an amino acid substitution from isoleucine (ATT) to methionine (ATG) in codon 132, which is located within the putative 5-phosphoribosyl-1-pyrophosphate (PRPP)-binding site of HPRT.