Structure and light-regulated expression of the gsa gene encoding the chlorophyll biosynthetic enzyme,glutamate 1-semialdehyde aminotransferase,in Chlamydomonas reinhardtii

The gsa gene, which encodes glutamate 1-semialdehyde (GSA) aminotransferase (GSAT), an enzyme in the chlorophyll and heme biosynthetic pathway, has been cloned from Chlamydomonas reinhardtii by complementation of an Escherichia coli hemL mutant. The deduced C. reinhardtii GSAT amino acid sequence has a high degree of similarity to GSAT sequences from barley, tobacco, soybean and various prokaryotic sources. In vitro enzyme activity assays from E. coli transformed with the C. reinhardtii GSAT cDNA showed that higher levels of GSAT activity are associated with the expression of the cDNA insert. Analysis of changes in mRNA levels in light:dark synchronized C. reinhardtii cultures was done by northern blotting. The level of GSAT mRNA nearly doubled during the first 0.5 h in the light and increased over 26-fold after 2 h in the light. This increase is comparable to previously reported increases in GSAT activity in dark-grown cultures transferred to the light, and is the first report of induction by light of a gene encoding an ALA biosynthetic enzyme in plant or algal cells. The accumulation of GSAT mRNA follows the pattern of chlorophyll accumulation and the pattern of chlorophyll a/b-binding protein (cabII-1) mRNA accumulation in these cells, suggesting that the two genes may be regulated by light through a common mechanism. Additional evidence that the GSAT mRNA may be transcriptionally regulated by light is found in the genomic sequence of the gsa gene. Two areas that are similar to sequences involved in the light regulation of genes from other organisms are located upstream of the GSAT-encoding region, and a third was detected internal to the coding region.     

The yeast protein Gcr1p binds to the PGK UAS and contributes to the activation of transcription of the PGK gene

Analysis of the upstream activation sequence (UAS) of the yeast phosphoglycerate kinase gene (PGK) has demonstrated that a number of sequence elements are involved in its activity and two of these sequences are bound by the multifunctional factors Rap1p and Abftp. In this report we show by in vivo footprinting that the regulatory factor encoded by GCR1 binds to two elements in the 3prime" align="BASELINE" border="0"> half of the PGK UAS. These elements contain the sequence CTTCC, which was previously suggested to be important for the activity of the PGK UAS and has been shown to be able to bind Gcrlp in vitro. Furthermore, we find that Gcr1p positively influences PGK transcription, although it is not responsible for the carbon source dependent regulation of PGK mRNA synthesis. In order to mediate its transcriptional influence we find that Gcrtp requires the Rap1p binding site, in addition to its own, but not the Abf1p site. As neither a Rapip nor a Gcr1p binding site alone is able to activate transcription, we propose that Gcr1p and Rapip interact in an interdependent fashion to activate PGK transcription.Communicated by C. P. Hollenberg     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep p7542370tnq2/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and p7880/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-carbons in uniformly p>13p>Cp>15p>N-labeled proteins. In-phase p>13p>Cp>p7880/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">p> magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from p>13p>Cp>p7880/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">p> to p>1p>Hp>p7880/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">p> leads to E.COSY-type cross peaks, from which the p>3p>J_H p>p7880/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">p> _co coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

非同位素PCR－单链构象多态性技术的建立和应用

ＰＣＲ－单链构象多态性技术问世以来,成为研究基因突变的工具,特别是在分子肿瘤学研究中,广泛应用于癌基因,抑癌基因突变的研究,常规ＰＣＲ－ＳＳＣＰ采用同位素标记ＰＣＲ产物,测序板电泳分离突变,在操作和费用上有种种局限,文章建立了一种非同位素ＰＣＲ－ＳＳＣＰ技术;通过不对称ＰＣＲ获得单链,普通ＰＡＧＥ分离,经银染检出突变,用这种方法,还研究了四株鼻咽癌细胞株ＣＮＥ１,ＣＮＥ２,ＨＫ１和ＳＵＮＥ１中肿瘤     

P53，Rb和c－myc基因在人脑原发性肿瘤中的转录表达

采用RNA斑点杂交分析,对21例人脑原发性胶质瘤和11例人脑膜瘤中p53,Rb和c-myc基因转录水平的表达进行研究.发现48.4%的肿瘤中p53基因表达减弱,21.9%的肿瘤中Rb基因表达减弱;71.9%的肿瘤中c-myc基因表达增强.在p53基因表达减弱的15例病例中有13例(80%)c-myc基因表达增强.结果表明,p53基因表达减弱和c-myc基因表达增强与人脑原发性肿瘤的发生有关.     

Genetic analysis of human p34CDC2 function in fission yeast

The p34p>cdc2p> protein kinase plays a key role in the control of the mitotic cell cycle of fission yeast, being required for both entry into S-phase and for entry into mitosis in the mitotic cell cycle, as well as for the initiation of the second meiotic nuclear division. In recent years, structural and functional homologues of p34p>cdc2p>, as well as several of the proteins that interact with and regulate p34p>cdc2p> function in fission yeast, have been identified in a wide range of higher eukaryotic cell types, suggesting that the control mechanisms uncovered in this simple eukaryote are likely to be well conserved across evolution. Here we describe the construction and characterisation of a fission yeast strain in which the endogenous p34p>cdc2p> protein is entirely absent and is replaced by its human functional homologue p34p>CDC2p>, We have used this strain to analyse aspects of the function of the human p34p>CDC2p> protein genetically. We show that the function of the human p34p>CDC2p> protein in fission yeast cells is dependent upon the action of the protein tyrosine phosphatase p80p>cdc25p> that it responds to altered levels of both the mitotic inhibitor p107p>2331p> and the p34p>cdc2p>-binding protein p13p>suc1p>, and is lethal in combination with the mutant B-type cyclin p56p>cdc13-117p>. In addition, we demonstrate that the human p34p>CDC2p> protein is proficient for fission yeast meiosis, and examine the behaviour of two mutant p34p>CDC2p> proteins in fission yeast.     

A break in the nitrogen cycle in aridlands? Evidence from δp15N of soils

We examined the content and isotopic composition of nitrogen within soils of a juniper woodland and found that a cryptobiotic crust composed of cyanobacteria, lichens, and mosses was the predominant source of nitrogen for this ecosystem. Disturbance of the crust has resulted in considerable spatial variability in soil nitrogen content and isotopic composition; intercanopy soils were significantly depleted in nitrogen and had greater abundance of p>15p>N compared to intra-canopy soils. Variations in the p>15p>N/p>14p>N ratio for inter- and intra-canopy locations followed similar Rayleigh distillation curves, indicating that the greater p>15p>N/p>14p>N ratios for inter-canopy soils were due to relatively greater net nitrogen loss. Coverage of cryptobiotic crusts has been reduced by anthropogenic activities during the past century, and our results suggest that destruction of the cryptobiotic crust may ultimately result in ecosystem degradation through elimination of the predominant source of nitrogen input.     

Genes of the R-phycocyanin II locus of marine Synechococcus spp., and comparison of protein-chromophore interactions in phycocyanins differing in bilin composition

R-phycocyanin II (RPCII) is a recently discovered member of the phycocyanin family of photosynthetic light-harvesting proteins. Genes encoding the p7376p817147/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0"> and p7376p817147/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0"> subunits of RPCII were cloned and sequenced from marine Synechococcus sp. strains WH8020 and WH8103. The deduced amino acid sequences of RPCII were compared to two other types of phycocyanin, C-phycocyanin (CPC) and phycoerythrocyanin (PEC). These three types vary in the composition of their covalently bound bilin prosthetic groups. In terms of amino acid sequence identity RPCII is highly homologous to CPC and PEC, suggesting that the known three-dimensional structures of the latter two are representative of RPCII. Thus the amino acid residues contacting the three bilins of RPCII could be inferred and compared to those in CPC and PEC. Certain residues were identified among the three phycocyanins as possibly correlating with specific bilin isomers. In overall sequence RPCII and CPC are more homologous to one another than either is to PEC. This probably reflects functional homology in the roles of RPCII and CPC in the transfer of light energy to the core of the phycobilisome, a function not attributed to PEC. The genomes of Synechococcus sp. strains WH8020, WH8103 and WH7803 share homologous open reading frames in the vicinity of RPCII genes. The nucleotide sequence extending 3p7376p817147/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> from RPCII genes in strain WH8020 revealed two open reading frames homologous to components of an p7376p817147/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">CPC phycocyanobilin lyase. These open reading frames may encode a lyase specific for the attachment of phycoerythrobilin to p7376p817147/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">RPCII.