Molecular diversity of L-type calcium channels. Evidence for alternative splicing of the transcripts of three non-allelic genes

The diversity of L-type calcium channels was probed using the polymerase chain reaction and primers based on regions conserved in the L-type skeletal muscle (CaCh 1) and cardiac calcium channels (CaCh 2). Related sequences were amplified from human heart, hamster heart, rabbit heart, mouse ovary, mouse BC3H1 cells, and hamster insulin-secreting (HIT) cells. Sequencing of various clones revealed the presence of alternate splicing in gene products coding for CaCh 1, CaCh 2, and a related calcium channel. This related gene product, which we refer to as neuroendocrine or CaCh 3, is expressed in brain and endocrine cells. The diverse products can be explained by the use of alternate exons of equal size, which account for changes in amino acid composition, in combination with an alternate splice acceptor site or an exon skipping event, which produces channels of variable length. Four variants were defined for the gene 3 product, subtypes 3a, 3b, 3c, and 3d that differed in both the sequence of the third membrane spanning segment of the fourth repeat unit (IVS3) and in the size of the linker between this and the fourth membrane spanning segment (IVS4). Three CaCh 2 variants were cloned, subtypes 2a, 2c, and 2d, that are homologous to the a, c, and d variants of CaCh 3. For the skeletal muscle calcium channel only two variants were isolated. They are homologous to those of the a and c subtypes of CaCh 2 or 3, in that they differ only in the size of the IVS3 to IVS4 linker. These results demonstrate that calcium channel diversity is created by both the expression of distinct genes and the alternate splicing of these genes.     

Structure and assembly of turnip crinkle virus. VI. Identification of coat protein binding sites on the RNA

Structural studies of turnip crinkle virus have been extended to include the identification of high-affinity coat protein binding sites on the RNA genome. Virus was dissociated at elevated pH and ionic strength, and a ribonucleoprotein complex (rp-complex) was isolated by chromatography on Sephacryl S-200. Genomic RNA fragments in the rp-complex, resistant to RNase A and RNase T1 digestion and associated with tightly bound coat protein subunits, were isolated using coat-protein-specific antibodies. The identity of the protected fragments was determined by direct RNA sequencing. These approaches allowed us to study the specific RNA-protein interactions in the rp-complex obtained from dissociated virus particles. The location of one protected fragment downstream from the amber terminator codon in the first and largest of the three viral open reading frames suggests that the coat protein may play a role in the regulation of the expression of the polymerase gene. We have also identified an additional cluster of T1-protected fragments in the region of the coat protein gene that may represent further high-affinity sites involved in assembly recognition.     

Arachidonic acid epoxygenase: structural characterization and quantification of epoxyeicosatrienoates in plasma.

Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase.     

Common denominators in the etiology and pathology of visceral lesions of cystic fibrosis and Keshan disease

The common denominator of a unique disseminated multi-focal milliary myocardial hyaline necrosis and fibrosis in Keshan disease (KSD) and cystic fibrosis (CF) and a commonality of the affected age groups of fetuses and preschool children led to the review of existing KSD autopsy material to search for pancreatic and hepatic lesions considered pathognomonic for CF. Pancreatic lesions considered pathognomonic for CF were found in 595, or 35% of 1700 documented cases of KSD. The pancreatic lesions were limited to tissues of fetuses and preschool children. Adults dying of KSD had diagnostic lesions limited to the cardiovascular system, liver, and skeletal muscle. Varying degrees of focal biliary cirrhosis were identified in 850, or 50% of the KSD autopsies, and 85, or 5% developed severe lobular cirrhosis. The common denominator in CF and KSD appears to be a primary or induced secondary selenium deficiency in age-susceptable humans, prenatally at or around 22 wk of fetal life, during early postnatal life, or during the rapid-growth preschool years. The basic difference between the natural history of CF and KSD is that the selenium deficiency is totally environmental in KSD and appears to be the result of a maternal malabsorptive syndrome or an abnormality of selenium transfer in CF.     

MNNG对细胞DNA合成及其分布的影响

本文用流式细胞光度术(FCM)等方法研究了MNNG,ENNG和DMS对HeLa细胞DNA含量分布的影响。经MNNG(6.8μmol/L)处理后,细胞分裂减少,DNA合成速率下降,S期细胞的比例随处理时间的延长而增加。DMS显示有类似的现象而ENNG的效应则较小。     

质粒YRP7的基本特性鉴定

质粒YRP7用氯霉素法扩增,碱变性裂解法提取,酸酚法及核糖核酸酶纯化后,得到了高产量(5.6mg/L培养液),高纯度(A260:A280=2.0)的质粒制品,经转化实验及酶切分析确定YRP7具有下列特征:大小为5.41±0.10kb,可赋予宿主细胞AmP~r、Tet~r的表型,对大肠杆菌C600的转化频率为10~(-6)、转化效率为1.5×10~6转化子/mgDNA。限制性内切酶BamH Ⅰ、ECoRⅠ、Hind Ⅲ及PstⅠ在其分子上的切点数分别为1、2、2、2,并确定了各酶切片段的分子大小,对BanHⅠ的单切点,经插入失活法证实其位于Tet~r的基因上。由上述特征可确定,质粒YRP7是一个比较理想的克隆载体。     

Ferritin gene organization: Differences between plants and animals suggest possible kingdom-specific selective constraints

Ferritin, a protein widespread in nature, concentrates iron ∼10¹¹–10¹²-fold above the solubility within a spherical shell of 24 subunits; it derives in plants and animals from a common ancestor (based on sequence) but displays a cytoplasmic location in animals compared to the plastid in contemporary plants. Ferritin gene regulation in plants and animals is altered by development, hormones, and excess iron; iron signals target DNA in plants but mRNA in animals. Evolution has thus conserved the two end points of ferritin gene expression, the physiological signals and the protein structure, while allowing some divergence of the genetic mechanisms. Comparison of ferritin gene organization in plants and animals, made possible by the cloning of a dicot (soybean) ferritin gene presented here and the recent cloning of two monocot (maize) ferritin genes, shows evolutionary divergence in ferritin gene organization between plants and animals but conservation among plants or among animals; divergence in the genetic mechanism for iron regulation is reflected by the absence in all three plant genes of the IRE, a highly conserved, noncoding sequence in vertebrate animal ferritin mRNA. In plant ferritin genes, the number of introns (n= 7) is higher than in animals (n= 3). Second, no intron positions are conserved when ferritin genes of plants and animals are compared, although all ferritin gene introns are in the coding region; within kingdoms, the intron positions in ferritin genes are conserved. Finally, secondary protein structure has no apparent relationship to intron/exon boundaries in plant ferritin genes, whereas in animal ferritin genes the correspondence is high. The structural differences in introns/exons among phylogenetically related ferritin coding sequences and the high conservation of the gene structure within plant or animal kingdoms suggest that kingdom-specific functional constraints may exist to maintain a particular intron/exon pattern within ferritin genes. In the case of plants, where ferritin gene intron placement is unrelated to triplet codons or protein structure, and where ferritin is targeted to the plastid, the selection pressure on gene organization may relate to RNA function and plastid/nuclear signaling. Received: 25 July 1995 / Accepted: 3 October 1995     

反义寡核苷酸片段可抑制鲫鱼视网膜电压依赖性钾离子通道的表达

爪蟾卵母细胞经注射鲫鱼视网总ＲＮＡ后,可表达大量电压依赖性钾离子通道。在此基础上,我们进一步发现,一个特定序列的寡核苷酸能专一地抑制该通道的表达。由于我们设计与合成的该片段与果蝇、小鼠中已克隆的钾通道中编码Ｎ端的一个多肽的ｍＲＮＡ完全互补,因此推测：鲫鱼视网膜中的Ｋ这个区域与其它物种的Ｋ通道有高度同源性,这为克隆该基因和研究它的功能提供了重要资料。     

团藻目新资料

本文报道湖北省武汉市团藻目7个属的5个新种,2个新变种,2个中国新记录。