Cloning and sequencing of the genes encoding the large and small subunits of the periplasmic (NiFeSe) hydrogenase of Desulfovibrio baculatus.

The genes coding for the large and small subunits of the periplasmic hydrogenase from Desulfovibrio baculatus have been cloned and sequenced. The genes are arranged in an operon with the small subunit gene preceding the large subunit gene. The small subunit gene codes for a 32 amino acid leader sequence supporting the periplasmic localization of the protein, however no ferredoxin-like or other characteristic iron-sulfur coordination sites were observed. The periplasmic hydrogenases from D. baculatus (an NiFeSe protein) and D. vulgaris (an Fe protein) exhibit no homology suggesting that they are structurally different, unrelated entities.     

Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources

Summary The primary structures of 11 proteins of ADP-glucose pyrophosphorylase are aligned and compared for relationships among them. These comparisons indicate that many domains are retained in the proteins from both the enteric bacteria and the proteins from angiosperm plants. The proteins from angiosperm plants show two main groups, with one of the main groups demonstrating two subgroups. The two main groups of angiosperm plant proteins are based upon the two subunits of the enzyme, whereas the subgroups of the large subunit group are based upon the tissue in which the particular gene had been expressed. Additionally, the small subunit group shows a slight but distinct division into a grouping based upon whether the protein is from a monocot or dicot source. Previous structure-function studies with the Escherichia coli enzyme have identified regions of the primary structure associated with the substrate binding site, the allosteric activator binding site, and the allosteric inhibitor binding site. There is conservation of the primary structure of the polypeptides for the substrate binding site and the allosteric activator binding site. The nucleotide sequences of the coding regions of the genes of 11 of these proteins are compared for relationships among them. This analysis indicates that the protein for the small subunit has been subject to greater selective pressure to retain a particular primary structure. Also, the coding region of the precursor gene for the small subunit diverged from the coding region of the precursor gene for the large subunits slightly prior to the divergence of the two coding regions of the genes for the two tissue-specific large subunit genes.Offprint requests to: J. Preiss     

A low-starch barley mutant,risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase,reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Ris? 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Ris? 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.     

Ribosomal and protein coding gene based multigene phylogeny on the family Streptomycetaceae

The phylogenetic relationship among the three genera of the family Streptomycetaceae was examined using the small and large subunit ribosomal RNA genes, and the gyrB, rpoB, trpB, atpD and recA genes. The total stretches of the analyzed ribosomal genes were 4.2kb, and those of five protein coding genes were 4.5 kb. The resultant phylogenetic trees confirmed that each genus formed an independent clade in the majority of cases. The G+C contents of rRNA genes were 56.9-58.9 mol%, and those of protein coding genes were 65.4-72.4 mol%, the latter being closer to those of the genomic DNAs. The average nucleotide sequence identity between the organisms were 94.1-96.4% for rRNA genes and 85.7-90.6% for protein coding genes, thus indicating that protein coding genes can give higher resolution than rRNA genes. In addition, the protein coding gene trees were more stable than the rRNA gene trees, supported by higher bootstrap values and other treeing algorithms. Moreover, the genome data of six Streptomyces species indicated that many protein coding genes exhibited higher correlations with genome relatedness. The combined gene sequences were also shown to give a better resolution with higher stability than any single genes, though not necessarily more correlated with genome relatedness. It is evident from this study that the rRNA gene based phylogeny can be misleading, and also that protein coding genes have a number of advantages over the rRNA genes as the phylogenetic markers including a high correlation with the genome relatedness.     

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.     

Nucleotide sequence of cDNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase from maize

We have cloned a full length cDNA for the small subunit of ribulose-1,5-bisphosphate carboxylase from C4 monocot maize, determined the complete nucleotide sequence of this cDNA and deduced its amino acid sequence. The cDNA insert included 513 bp of the coding region, and 65 and 252 nucleotides of the 5' and 3' untranslated regions, respectively. The transit and mature peptides have, respectively, 47 and 123 amino acids. Comparison with the small subunit genes from other plants revealed that the maize small subunit is similar to the wheat one, there being 73% homology between the transit peptides and 64% between the mature proteins. This indicates that there is no noteworthy difference between the C3 and C4 small subunit structures. Extreme codon bias was observed for this gene, and similar codon preferences are observed for other proteins highly expressed in maize leaf, light harvesting chlorophyll binding protein and phosphoenolpyruvate carboxylase. The results indicate that preferential codon usage for highly expressed genes occurs in maize leaf.     

Functional Interactions among Two Yeast Mitochondrial Ribosomal Proteins and an mRNA-Specific Translational Activator

Expression of the Saccharomyces cerevisiae mitochondrial gene coding cytochrome c oxidase subunit III is specifically activated at the level of translation by at least three nuclear genes, PET122, PET494 and PET54. We have shown previously that carboxy-terminal deletions of PET122 are allele-specifically suppressed by mutations in an unlinked nuclear gene, termed PET123, that encodes a small subunit ribosomal protein. Here we describe additional pet122 suppressors generated by mutations in a second gene which we show to be the previously identified nuclear gene MRP1. Like PET123, MRP1 encodes a component of the small subunit of mitochondrial ribosomes. Our mrp1 mutations are allele-specific suppressors of carboxyl-terminal truncations of the PET122 protein and do not bypass the requirement for residual function of PET122. None of our mrp1 mutations has an intrinsic phenotype in an otherwise wild-type background. However, some of the mrp1 mutations cause a non-conditional respiratory-defective phenotype in combination with certain pet123 alleles. This synthetic defective phenotype suggests that the ribosomal proteins PET123 and MRP1 interact functionally with each other. The fact that they can both mutate to suppress certain alleles of the mRNA-specific translational activator PET122 strongly suggests that the PET122 protein promotes translation of the coxIII mRNA via an interaction with the small subunit of mitochondrial ribosomes.     

G蛋白亚单位基因家族研究进展

G蛋白由α、β、γ三个亚单位组成异源三聚体。目前已发现16个α、6个β和12个γ基因。G蛋白亚单位基因家族相当保守并且原始,几乎所有G蛋白基因外显子-内含子连接均遵从GT-AG规则,并且各亚单位基因编码区内含子结构和位置显示出很高的保守性。多数G蛋白基因具有持家基因的特点。G蛋白基因在基因组中的分布存在着丛集的倾向,有5对α基因呈二联串连排列。     

Characterization of HMW prolamines and their coding sequences from Crithopsis delileana

The high-molecular-weight (HMW) prolamines subunits and their coding sequences from wheat-related diploid species Crithopsis delileana were investigated. Only one HMW prolamine subunit with the similar electrophoresis mobility to the y-type HMW glutenin subunit of hexaploid wheat was observed in two accessions of C. delileana by SDS-PAGE analyses of the total storage protein fractions. It was confirmed by sequencing and expression analysis that this prolamine subunit was an x-type subunit. The amino acid sequence of this subunit had the similar typical structure to those of x-type HMW glutenin genes previously described in wheat. An in-frame stop codon was found in the coding sequences of y-type prolamine subunits. It was found by specifically extraction of HMW prolamines and sequence analysis that the coding regions of Ky prolamine subunit gene is very likely to be not expressed as a full-length protein. Phylogenetic analysis indicated that the Kx subunit could be clustered together with 1Ax1 subunit by an interior paralleled branch, and Ky subunit (inactive) was most closely related to the 1Ay subunit. The coding sequences of Kx subunit could successfully be expressed in bacterial expression system, and the expressed protein had the same electrophoresis mobility as the Kx subunit from the seed of C. delileana. It was the first time that the HMW prolamines subunits encoded by K genome of C. delileana were characterized.     

The cytochrome C oxidase genes in blue-green algae and characteristics of the deduced protein sequence for subunit II of the thermophilic cyanobacterium Synechococcus vulcanus.

Blue-green algae (cyanobacteria) contain both primitive photosynthetic and respiratory systems in their membranes. The controversial genes coding for an alpha alpha 3-type cytochrome oxidase in cyanobacteria were examined. The DNA probe coding for the most conserved part of subunit I hybridized with DNA fragments from four cyanobacterial species. We have cloned the genes coding for subunits I and II from the genomic library of the thermophilic cyanobacterium Synechococcus vulcanus and determined the nucleotide sequence of the subunit II gene. The deduced protein sequence (327 amino acid residues) indicates that there are two hydrophobic segments near the N-terminus and a hydrophilic intermembrane domain containing ligands for CuA (the ESR-active Copper) similar to other subunit IIs. The S. vulcanus subunit II does not contain the cytochrome c moiety that is present in bacilli and thermophiles.     

Maize genes encoding the small subunit of ADP-glucose pyrophosphorylase

Plant ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme composed of two large and two small subunits. Here, we report the structures of the maize (Zea mays) genes encoding AGP small subunits of leaf and endosperm. Excluding exon 1, protein-encoding sequences of the two genes are nearly identical. Exon 1 coding sequences, however, possess no similarity. Introns are placed in identical positions and exhibit obvious sequence similarity. Size differences are primarily due to insertions and duplications, hallmarks of transposable element visitation. Comparison of the maize genes with other plant AGP small subunit genes leads to a number of noteworthy inferences concerning the evolution of these genes. The small subunit gene can be divided into two modules. One module, encompassing all coding information except that derived from exon 1, displays striking similarity among all genes. It is surprising that members from eudicots form one group, whereas those from cereals form a second group. This implies that the duplications giving rise to family members occurred at least twice and after the separation of eudicots and monocot cereals. One intron within this module may have had a transposon origin. A different evolutionary history is suggested for exon 1. These sequences define three distinct groups, two of which come from cereal seeds. This distinction likely has functional significance because cereal endosperm AGPs are cytosolic, whereas all other forms appear to be plastid localized. Finally, whereas barley (Hordeum vulgare) reportedly employs only one gene to encode the small subunit of the seed and leaf, maize utilizes the two genes described here.     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列.表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似.在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区.但是,本研究所克隆的沉默型1Av基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白.讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及lAy基因沉默的机制.     

野生大豆rbcS基因的克隆及结构分析

核酮糖１,５二磷酸羧化酶（Ｒｕｂｉｓｃｏ,Ｅ．Ｃ．４．１．１．３９）是光合碳代谢中的关键酶,也是植物中研究最为广泛深入的一种酶。高等植物的Ｒｕｂｉｓｃｏ大、小亚基分别由叶绿体和核基因组编码。迄今已有几十种光合生物的Ｒｕｂｉｓｃｏ大、小亚基的基因（ｒｂｃＬ、ｒｂｃＳ）结构得到阐明［１］。在高等植物中ｒｂｃＳ基因由多基因家族编码,结构较为复杂,但它同时又是一种相对保守的基因,且同一物种内各ｒｂｃＳ基因成员是协同进化的,因此ｒｂｃＳ基因适合于植物分子进化及系统分类的研究［２］。我国是栽培大豆（Ｇｌｙｃ…