An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis.

The three chromosomal DNAs of S. pombe have been fractionated by pulsed field gel electrophoresis. The resulting molecular karyotype will greatly speed gene mapping in this organism, and it indicates that the separation range of the technique extends to DNA molecules as large as 9,000,000 base pairs.     

Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination

The early events in site-specific excisive recombination were studied with phage lambda half-att sites that have no DNA to one side of the strand exchange region; they carry a single core-type integrase binding site and either P or P' arm flanking DNA. These half-attR and half-attL sites exhibit normal properties for the initial (covalent) top-strand transfer and form stable intermediates independent of later steps in the reaction. With these novel substrates we show that Xis specifically promotes the first strand exchange and that attL enhances Int cleavage at the top-strand site of attR. It is also shown that synapsis and initial strand transfers do not require DNA-DNA pairing but are mediated by protein-protein and protein-DNA interactions. These involve the two top-strand Int binding sites (required for the first strand exchange) and, in addition, one of the two bottom-strand sites (C') responsible for the second strand exchange.     

Effect of volume expansion on hemodynamic variables in nephrectomized dogs

We have previously demonstrated that blood pressure elevation by acute blood volume expansion is volume-dependent during the infusion period and resistance-dependent in the post-infusion period in normal anesthetized dogs, and that such an increase in blood pressure is associated with a potentiation of the pressor response to norepinephrine. To evaluate the possible renal contribution to these hemodynamic changes, blood volume expansion was performed for 1 h with dextran dissolved in lactated Ringer's solution (20 ml/kg) in 15 nephrectomized dogs. The mean blood pressure, cardiac output and total peripheral resistance at the end of infusion were 126%, 225% and 60%, respectively; 3 h after volume expansion they were 126%, 151%, and 92% respectively. However, in 4 dogs, there was an increase in mean blood pressure (138%) 3 h after volume expansion. This was thought to result from an increase in the total peripheral resistance (133%) associated with the recovery of cardiac output (106%). The pressor response to norepinephrine (0.5 microgram/kg) was potentiated after volume expansion. These results indicate that the handling of volume by the kidney contributed to the maintenance of an elevated level of cardiac output. However, nephrectomy did not seem to interfere with the hemodynamic switching of the causative factor for blood pressure elevation from increased cardiac output to increased total peripheral resistance. Neither was the potentiation of pressor response to norepinephrine affected.     

Characterization of Schizosaccharomyces pombe minichromosome deletion derivatives and a functional allocation of their centromere.

A 530 kb long Schizosaccharomyces pombe linear minichromosome, Ch16, containing a centric region of chromosome III, has previously been made. In the present study, we constructed a number of deletions in the right and/or left arms of Ch16, and compared their structure and behaviour with Ch16. The functional centromere, cen3, is allocated within a 120 kb long region which is covered by the shortest derivative, Ch10, and is comprised mostly of centromeric repeating sequences. The shortest minichromosome is stable in mitosis and the copy number control is apparently precise. In monosomic meiosis it segregates normally. In disomic meioses, however, the frequency of non-disjunction is very high, suggesting that it may not form a pair. The mitotic loss rate of one of the left-arm deletions, ChR32, which lacks a part of the centromeric repeating sequence, is the highest of all the deletions. This deletion also exhibits the highest precocious sister chromatid separation in meiosis I, suggesting that sister chromatid association might become weakened in ChR32. Our results indicate that the proper meiotic segregation of S.pombe minichromosomes is dependent upon the formation of a bivalent. S.pombe may not have the 'distributive segregation' found with Saccharomyces cerevisiae minichromosomes.     

Effect of chemical modifications on freeze denaturation of lactate dehydrogenase

Lactate dehydrogenase (LDH) was chemically ethyl-acetimidated (EA-), dimethyladipimidated (DMA-), carbamylated, acetylated, acetoacetylated, or succinylated in order to alter the ionic charges on the epsilon-amino group of lysine residues. Acetylation, acetoacetylation, and succinylation, which change the positive charge at the lysine side chains to a negative one, inactivated the enzymic activity, but the rest of the modifications exerted no such inactivating effects. The active modified enzymes were subjected to freeze denaturation study, using the enzymic activity as an indication of the degree of the denaturation. The active enzymes were diluted with deionized water and stored in a freezer (-23 degrees C) for 1-3 days. Enzymic activity was assayed immediately after thawing. All the modified enzymes retained their activity even after the 3-day frozen storage, while the control or native enzyme lost its activity within 1 day of storage. Furthermore, the modified LDHs freeze-stored in 0.2 M monosodium glutamate (MSG) or 0.2 M lysine-hydrochloride (Lys-HCl) retained their activity. The cryoprotective effects exerted by the modifications and by 0.2 M MSG seemed to be synergistic, whereas those exerted by the modifications and by 0.2 M Lys-HCl did not. The mechanisms of cryoprotection and freeze denaturation are discussed in relationship with the cryoprotective effect exerted by already known cryoprotectants, such as sucrose or dimethyl sulfoxide.     

Roles of prepubertal androgen, estrogen or androgen plus prolactin on androgen-induced proliferative response of seminal vesicles in adult mice

Male mice castrated on day 0 after birth were pretreated daily with testosterone propionate (TP, 4 micrograms/g body weight), 17 beta-estradiol (E2, 0.2 micrograms/g body weight) or vehicle for 21 days starting from day 20. In another experiment, male mice were castrated on day 25; two pituitaries from 60-day-old females were immediately grafted under the capsule of the left kidney in one group. The castrated mice with or without grafts were pretreated daily with TP (4 or 20 micrograms/g body weight) for 36 days starting from day 25, and the left kidney was removed on day 60. Daily TP injections (4 micrograms/g body weight) were started again at 30 days after the end of pretreatments to examine androgen-induced proliferation, and incorporation of 5-[125I]iodo-2'-deoxyuridine into the whole seminal vesicles was used as an index of proliferation. In the neonatally castrated mice, both TP and E2 pretreatments given during the prepubertal period significantly increased seminal vesicle weight even long after the end of the pretreatments. However, androgen-induced proliferative response found in the neonatally castrated adult mice (poor response; long duration with a low peak) was changed to that found in mice castrated at adulthood (good response; short duration with a high peak) by the TP pretreatment only but not at all by the E2 pretreatment. In the mice castrated on day 25, a pharmacological dose of TP or TP plus hyperprolactin could not enhance or change the adult castration type of androgen-induced proliferation induced by physiological prepubertal androgens, although both treatments significantly enhanced the prepubertal growth of the seminal vesicles.     

Synthesis of the vasoconstrictor peptide endothelin in kidney cells

The expression plasmid containing human prepro-endothelin cDNA was constructed and introduced into COS-7 cells. Mature endthelin, consisting of 21 amino acid residues, was secreted into the culture medium of the transfected cells and was also synthesized by non-transfected COS-7 cells. Normal kidney cells derived from other species also synthesized and secreted endothelin. Partial characterization of endothelins produced by kidney cells suggested that existence of new types of endothelin. This is the first report of the vasoconstrictor peptide endothelin being synthesized in kidney cells.     

Determination of guanine by high-performance liquid chromatography with electrochemical detection: application to DNA and RNA assays

A highly sensitive assay for guanine was developed using high-performance liquid chromatography with electrochemical detection (ECD). Guanine was susceptible to the electrochemical oxidation, and ECD response was proportional to the amount of guanine in the range 0.25-4 pmol of guanine. The ECD of guanine was applicable to the analysis of nucleic acids. DNA and RNA were hydrolyzed in 0.03 and 3 M HCl, respectively, and guanine liberated from the nucleic acids was separated on a reverse-phase column and determined by ECD. The method allowed detection of 0.2 ng of calf thymus DNA or tRNA. An application of the method is shown for DNA and RNA assays in trichloroacetic acid extracts of rat adrenal and liver.     

Regional distribution of DNA and RNA in rat brain: A sensitive determination using high-performance liquid chromatography with electrochemical detection

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.     

Holocene sedimentary history of some coastal plains in Hokkaido,Japan IV. Diatom assemblages in the sediments from Kushiro moor (2)

Diatom assemblages of sediments obtained from three sites on Kushiro Moor were analyzed to investigate the Holocene sedimentary history. The results showed that: 1) The Takkobu site was originally at the bottom of the paleo-Kushiro Bay, and after-wards the paleo-Takkobu Lagoon developed, became sealed off, and changed to a freshwater lake. The succession to peat moor probably began about 2000 yr B.P. at the Takkobu site. 2) The Tsurui site was originally at the bottom of the paleo-Kushiro Bay, then changed to the paleo-Kushiro Lagoon and became peat moor as a result of the first Holocene regression, which finished about 3600 yr B.P. The site then returned to a brackish lake again, probably due to the second Holocene transgression between 3600 and 3000 yr B.P., thereafter passing through brackish lake and freshwater lake stages, and eventually becaming peat moor at about 2000 yr B.P., 3) At the Chuo site, the second paleo-Kushiro Bay developed again as a result of the second Holocene transgression, which finished about 3000 yr B.P. Thereafter, brackish or freshwater lakes, rivers, and then peat moor developed in the central area of Kushiro Moor. 4) The second marine diatom zone (MD₂ Zone), which indicates the second Holocene transgression, complete by about 3000 yr B.P., is detected only at the Chuo site in the central area of Kushiro Moor.