植物同工酶的研究和应用

同工酶作为生物界存在的一种普遍现象受到了广泛的研究。同工酶学(Isozymology)虽然不是一个确定的学科,但它已渗透到生物学的每一个领域,尤其是对酶学、物理生物化学和比较生物化学、生理学、发育生物学、细胞和分子生物学、遗传学、进化论等学科更加完善起了并将继续起着重大作用。在科学史上,很少有象同工酶的概念及     

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.The authors contributed equally to this work     

Purification and characterization of a truncatedBacillus subtilis α-amylase produced byEscherichia coli

ABacillus subtilis amylase gene was inserted into a plasmid which transferred toEscherichia coli. During cloning, a 3 region encoding 171 carboxyterminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of theB. subtilis complete -amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with aK _m of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50°C, and the purified enzyme was stable at temperatures up to 50°C. While Hg²⁺, Fe³⁺ and Al³⁺ were effective in inhibiting the truncated enzyme Mn²⁺ and Co²⁺ considerably enhanced the activity.     

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented byHLA-B7 and is encoded by a new gene that we have namedRAGE1. No expression ofRAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.     

Analysis of the biosynthetic process of cellulose and curdlan using C-labeled glucoses

In order to elucidate the biosynthetic process of cellulose and curdlan, ¹³C-labeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 31749), from culture media containing -(1-¹³C)glucose, -(2-¹³C)glucose, -(4-¹³C)glucose, or -(6-¹³C)glucose as the carbon source, and their structures were determined by ¹³C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-1, C-3 and C-5, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-1 and C-3, from C-4 to C-1, C-2 and C-3, and from C-6 to C-1 and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-1 to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

Species richness and endemism of birds in Bioko

The status of 50 taxonomically unique bird taxa found in Bioko is described. A complete updated species list for birds in Bioko is also given. Notes on habitat occupied and altitudinal distribution of each species as well as information on their general abundance is also included. Species distributions may have been affected by habitat modifications brought about by humans but most taxonomically unique forms are found in the montane areas. Comparisons are made with the neighbouring Mount Cameroon area.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Aurintricarboxylic Acid Protects Hippocampal Neurons from NMDA-and Ischemia-Induced Toxicity In Vivo

Abstract: The polymeric dye aurintricarboxylic acid (ATA) has been shown to protect various cell types from apoptotic cell death, reportedly through inhibition of a calcium-dependent endonuclease activity. Recent studies have indicated that there may be some commonalities among apoptosis, programmed cell death, and certain other forms of neuronal death. To begin to explore the possibility of common biochemical mechanisms underlying ischemia-or excitotoxin-induced neuronal death and apoptosis in vivo, gerbils or rats subjected to transient global ischemia or NMDA microinjection, respectively, received a simultaneous intracerebral infusion of ATA or vehicle. As a biochemical marker of neuronal death, spectrin proteolysis, which is mediated by activation of calpain I, was measured in hippocampus after 24 h. ATA treatment resulted in a profound reduction of both NMDA-and ischemia-induced spectrin proteolysis, consistent with the possibility of some common mechanism in apoptosis and other forms of neuronal death in vivo.     

A novel tenascin type III repeat is part of a complex of tenascin mRNA alternative splices.

Sequence analysis of two human tenascin encoding cDNA clones from a cDNA library of the U251 glioblastoma cell line revealed the presence of a novel 276 bp tenascin type III fibronectin like repeat. This alternatively spliced type III repeat designated AD1 is located between the previously identified repeats 10 and 11 and has sequence homology with human, chicken and mouse tenascin type III repeats. These results show that tenascin has at least 16 consecutive fibronectin like type III repeats. PCR amplification of random primed mRNA with specific type III repeat primers revealed a pattern of multiple alternative splices of AD1 and flanking type III repeats. The alternative splice variants were confirmed by direct sequencing. Differences were observed in the expression of the various alternative splices of tenascin mRNA between tumor and normal cells and may thus indicate differences in tenascin isoform expression and function in normal and tumor cells. PCR and Southern analysis of genomic DNA indicate that AD1 is coded by a single exon present in both human and mouse genome.