Differential effects of cisplatin on mouse hepatic and renal mitochrondrial DNA

The objective of this study was to determine if the nephrotoxic effects induced by cisplatin were correlated to mitochondrial DNA damage. Comparisons were made with the liver since hepatotoxicity is rarely observed. Cisplatin doses of 10, 20 and 40 mg/kg were administered intraperitoneally to C57BL/6J mice. Mitochrondrial DNA was isolated from both the hepatic and renal tissues and quantitated by hybridization with a specific mitochondrial probe. Cisplatin caused differential effects on mouse hepatic and renal mitochondrial DNA. The 10 and 20 mg/kg dose caused an elevation in mitochondrial DNA levels in the hepatic, but no increase in the renal tissue was observed. This is the first study demonstrating an organ specific effect of cisplatin at the DNA level.     

Stimulation and inhibition of cardiac myocyte proliferation in vitro

Summary We have examined the effect of crude cardiac tissue extracts as well as that of several growth factors and triiodothyronin (T3) on DNA synthesis of cardiac myocytes in culture. Extracts from embryonic and adult cardiac tissue stimulated DNA synthesis of myocytes. Atrial myocytes exhibited overall higher degree of stimulation than their ventricular counterparts and extracts from adult atrial tissue had the highest apparent mitogenic activity for atrial myocytes. We have shown that adult heart contains basic fibroblast growth factor (bFGF), especially in the atria [1]. Transforming growth factor (TGF) and insulin-like growth factors (IGFs) are also accumulated in cardiac tissues [2, 3]. We found that bFGF and the IGFs stimulate myocyte cell proliferation and DNA synthesis. These factors also stimulate cardiac non-muscle proliferation, especially in the presence of serum. TGF inhibited proliferation and DNA synthesis and cancelled the effect of bFGF or IGFs on the myocytes. T3 also diminished the bFGF-induced mitogenic stimulation of cardiomyocytes. Our data suggest that these factors may be involved in the regulation of cardiomyocyte proliferation in vivo.Abbreviations bFGF basic Fibroblast Growth Factor - BSA Bovine Serum Albumin - DM Defined Medium - Fes Fetal calf serum - FITC Fluorescein - IGF Insulin-like Growth Factor - IgG Immunoglobulin - LI Labeling Index - PBS Phosphate Buffered Saline - T3 Triiodothyronine - TGF Transforming Growth Factor      

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA     

The Drosophila PROS-28.1 gene is a member of the proteasome gene family

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-M_r complex possessing proteolytic activity. Screening a Drosophila λgt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

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.     

A rapid miniprep for the isolation of total DNA fromAgrobacterium tumefaciens

A procedure which avoids the use of phenol-chloroform and RNAase for the isolation of total DNA fromA. tumefaciens is described. Specific precipitation of protein by 2.5 M ammonium acetate is employed and much of the RNA is removed by an isopropanol precipition step. The procedure yields easily restrictable, good quality DNA and is probably applicable to other Gramnegative bacteria.     

Non-radioactive detection of single-copy DNA-DNA hybrids

A new procedure for non-radioactive detection of single-copy DNA-DNA hybrids combines an existing non-radioactive labeling and detection kit with a new substrate AMPPD for the enzyme alkaline phosphatase. The main advantages of this procedure are the possibility to reuse the blots easily and the much shorter detection time compared to radioactive detection methods.     

人体小卫星DNA探针的制备

根据人体小卫星DNA核心顺序,化学合成长23碱基寡核苷酸探针,筛选人体基因组文库,旨在获得能用作遗传分析探针的小卫星顺序。结果得到15个含小卫星的阳性重组子。随机取其一(C_(35.9))作探针,试做群体分析。所有个体均可检出多条杂交带。其中某些带具有多态性。在一定检测条件下,检出的DNA图谱在有限的个体内具有个体特异性。结果表明筛选文库得到的小卫星顺序可用于小卫星多态性的检测。其它小卫星探针的筛选和应用性研究正在进行。     

Ribosomal DNA non-transcribed spacer polymorphism in subterranean mole rats: genetic differentiation,environmental correlates and phylogenetic relationships

Summary An analysis is presented of genetic differentiation in the non-transcribed spacers of ribosomal DNA (NTS rDNA). Diversity, environmental correlates and the phylogenetic relationships are examined within and between species of the actively speciating subterranean mole rat, superspeciesSpalax ehrenbergi (2n=52, 54, 58, 60) in Israel. This analysis is based on a previous study of the geographic distribution of restriction fragment length polymorphisms of NTS rDNA. Here we present results indicating that NTS rDNA diversity exists mostly (66%) within populations, while 20% is between populations within species, and 14% between species. Multivariate discriminant analysis succeeded in separating 10 of the 13 populations (77%) into their correct chromosomal species, on the basis of the combination of three NTS rDNA repetypes. The phylogenetic relationships suggest that the complex involves two pairs of closely related species (2n=52–54 and 2n=58–60). NTS rDNA diversity, as well as the decrease southward in frequency of repetype C, are correlated with climatic factors of humidity and temperature. These data are discussed in terms of the evolutionary forces of migration and selection which may cause NTS rDNA differentiation. Climatic selection appears to be the major differentiating factor of NTS rDNA.