Cloning and characterization of a tandemly repeated DNA sequence in the crane family (Gruidae)

A tandemly repeated DNA sequence possessing a unique PstI site has been characterized in several species of the crane family. The "Pst family" comprises at least 8800 monomer units 187 base pairs (bp) in length and constitutes 0.14% of the genome of the sarus crane (Grus antigone). The array is located in the centromeric heterochromatin of chromosome 2 in the two species where in situ hybridizations of a cloned monomer to metaphase chromosome spreads were carried out. DNA sequence comparisons between five monomer units from G. antigone revealed a high degree of homology between four of the individual repeats, while the fifth was somewhat divergent. The G + C content deduced from the DNA sequence makes it likely that the Pst family constitutes part of a density satellite seen in profiles of crane DNA centrifuged to equilibrium in CsCl. The common occurrence of tandem arrays such as the Pst family, with repeat lengths close to 200 bp, leads us to an hypothesis implicating nucleosomes in the evolution of such families.     

Individual rabbit cardiac myocytes have different thresholds for alpha myosin heavy chain regulation by thyroid hormone

Myocytes in adult rabbit ventricle express and alpha and a beta form of myosin heavy chain (MHC). The alpha-MHC distribution detected with indirect immunofluorescence has been found in different proportions in adjacent myocytes producing a mosaic staining pattern. The basis for cell-specific expression of the alpha-MHC isoform is not known. Since thyroid hormone is a major regulator of myosin gene expression, we varied the plasma thyroid level and followed the alpha-MHC content within a population of myocytes. Ventricular myocytes were induced to become 100% beta-MHC by placing the rabbits on a 0.15% propylthiouracil diet for 70 days. L-triiodothyronine (LT3) over a dose range of 1 to 10 micrograms/kg/day was delivered by an osmotic minipump for 5 days, with actual serum levels confirmed by LT3 radioimmunoassay to be in the range of from 115 to 1,230 ng/dl. The amount of alpha-MHC that returned was estimated in randomly selected cells by measuring the relative intensity of the fluorescence-tagged secondary antibody. The normal mosaic pattern of alpha-MHC expression in the left ventricle returned with an LT3 dose of 2-5 micrograms/kg/day. The first myocytes to express alpha-MHC were in the subepicardium and did so at a LT3 serum level of 115 of ng/dl. All myocytes of the ventricular wall expressed alpha-MHC at serum levels above 1,230 ng/dl. These data are interpreted to show that the variation of myosin isoform content seen in the adult heart is indicative of heterogeneity of thyroid sensitivity, with the threshold for serum LT3 being between 115 and 370 ng/dl.     

微管的冷稳定性与植物抗寒性关系的研究

利用间接免疫荧光的细胞化学技术对番茄、黄瓜、菠菜、甜菜及小麦等不同抗寒性植物微管的冷稳性进行了比较研究。结果指出,不抗寒的喜温性植物番茄和黄瓜的气孔保卫细胞的微管在0℃—1℃冷处理3小时即解聚;属于中等抗寒性植物的菠菜和甜菜幼苗经秋季低温锻炼后,其气孔保卫细胞的微管在0℃和—5℃低温处理3小时,均不发生解聚;具有较强抗寒性的冬小麦品种农大139幼苗在2—3℃低温锻炼期间,微管结构保持完整,经过15天低温锻炼的幼苗在-8℃冰冻处理3小时,微管也不受破坏。这些结果表明,微管的冷稳性与植物的抗寒性成正相关。     

Molecular cloning and sequencing of a cDNA encoding N alpha-acetyltransferase from Saccharomyces cerevisiae

Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and is catalyzed by an N alpha-acetyltransferase. Recently, a eukaryotic N alpha-acetyltransferase was purified to homogeneity from Saccharomyces cerevisiae, and its substrate specificity was partially characterized (Lee, F.-J. S., Lin L.-W., and Smith, J. A. (1988) J. Biol. Chem. 263, 14948-14955). This article describes the cloning from a yeast lambda gt11 cDNA library and sequencing of a full length cDNA encoding yeast N alpha-acetyltransferase. DNA blot hybridizations of genomic and chromosomal DNA reveal that the gene (so-called AAA1, amino-terminal, alpha-amino, acetyltransferase) is present as a single copy located on chromosome IV. The use of this cDNA will allow the molecular details of the role of N alpha-acetylation in the sorting and degradation of eukaryotic proteins to be determined.     

Purification, characterization, and amino terminal sequence of xylose reductase from Candida shehatae.

D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.     

Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.     

The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters.

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.     

On the sensitivity of intact cells to perturbation by ethanol

A comparison was made of ethanol's effects on the order of plasma membranes in intact cells and some isolated membrane preparations. Order was assessed by steady-state fluorescence polarization techniques using the non-permeant probe, TMA-DPH. The data show that two cultured cells, rat neonatal astroglial and N2A neuroblastoma, were sensitive to significant ethanol-induced disordering within the anesthetically relevant range (100 - 200 mM). Human erythrocytes, cultured fibroblasts and homogenized astroglial cells required higher ethanol concentrations (greater than 250 mM) to produce a similar effect. Intact erythrocytes were approximately twice as sensitive as erythrocyte ghost membranes to ethanol-induced perturbation. The neonatal glial and N2A cells were approximately five times more sensitive than synaptic membranes to ethanol effects. DMPC and DMPC + cholesterol liposomes and myelin membranes were insensitive to ethanol's effects. The incorporation of 10 mole % ganglioside GM1 sensitized the liposomes to ethanol-induced perturbation.     

Hypophysectomy decreases and growth hormone increases the turnover and mass of rat liver glutamine synthetase

Hypophysectomy diminishes rat liver glutamine synthetase (GS) activity and growth hormone (GH) administration restores this activity to normal levels; brain GS is unaffected. We have now investigated the effects of long-term hypophysectomy (45-day) and GH treatment on the GS mass (amount of enzyme) and turnover in rat liver and brain. Labeled GS was isolated by immunoprecipitation at intervals between one and six days after pulse administration of [U-14C] leucine and the GS half-life (t1/2) was determined. The GS mass was obtained by immunoassay and by calculation using the specific activity of purified GS. GS turnover was calculated by multiplying the GS mass by the first-order rate constant of degradation (kd). During the time course of each experiment, the GS mass did not change, indicating that in each of the three hormonal states studied, a steady state existed. Hypophysectomy increased the t1/2 of hepatic GS from 3.8 to 8.8 days and decreased GS turnover from 0.38 to 0.1 microgram/100 g body wt/day; the GH regimen used restored the turnover to above normal levels, 0.6 microgram/100 g body wt/day. The GS mass decreased from 2.0 to 1.2 micrograms/100 g body wt and GH restored the GS mass to normal levels. The brain enzyme was not affected by hypophysectomy or GH.     

Nucleotide sequence of cDNA for Acacia confusa trypsin inhibitor and implication of post-translation processing.

Synthetic oligonucleotides corresponding to all possible sequences of N-terminal and C-terminal region of Acacia confusa trypsin inhibitor were used to generate ACTI-related sequences using the polymerase chain reaction on the cDNAs encoding ACTI of the seeds of legume, A. confusa. The deduced amino acid sequence agreed with that determined by the peptide analysis except an extra amino acid residue, serine, was found at the junction of A and B chain, which was removed by post-translation processing with specific protease(s). The substrate specificity of the protease(s) was found to cleave at the C-terminal sites of asparagine and serine, which was also shown to be the same case for another plant protein, abrin, isolated from legume, Abrus precatorius.