Molecular cloning and chromosomal location of genes encoding the “Early-methionine-labelled” (Em) polypeptide of Triticum aestivum L. var. Chinese Spring

Summary The Early-methionine-labelled (Em) polypeptide is the most abundant cytosolic polypeptide found in mature wheat embryos. Using a near full-length cDNA clone as a hybridisation probe to detect genomic sequences by Southern blotting of electrophoretic separations of genomic DNA derived from Triticum aestivum L. var. Chinese Spring and a series of its aneuploid derivatives, we demonstrate that the Em polypeptide is the product of a small multigene family in which the copies are located on each of the long arms of the homoeologous group 1 chromosomes. Screening of a variety of genotypes additionally reveals a number of restriction fragment length polymorphisms associated with these loci. Screening of a library of genomic DNA cloned in the vector EMBL 4 has resulted in the isolation of a genomic fragment containing two closely linked Em genes. These are separated by ca. 2.5 kb. Analysis of restriction enzyme digests of this clones fragment has identified it as originating from chromosome 1A.     

Isolation and cDNA cloning of an Em-like protein from mung bean (Vigna radiata) axes

Until now no 'early-methionine-labelled' (Em) proteins have been reported in the Fabaceae. To check whether a previously isolated low-molecular mass albumin from dry mung bean embryonic axes possibly corresponded to an Em-like protein, the protein was purified, sequenced and its cDNA clone isolated and characterized. N-terminal sequencing of cyanogen bromide cleavage products of the protein revealed homology with previously described Em-like proteins from other species. Analysis of cDNA clones encoding the mung bean Em protein revealed the presence of two classes of Em proteins and confirmed their homology to the previously characterized Em-like proteins. In vivo labelling and northern blot analysis further demonstrated that the mung bean protein is synthesized during early germination of the axes and that abscisic acid (ABA) extends its synthesis. It appears, therefore, that legumes also contain maturation-specific, ABA-responsive Em-like proteins.     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.     

Modification of Lactobacillus beta-glucuronidase activity by random mutagenesis

The Lactobacillus gasseri ADH beta-glucuronidase gene, gusA, was cloned previously and found to exhibit excellent activity in acidic pH ranges, with maximal activity at pH 5.0. In contrast, activity was limited in neutral pH ranges of 6-7. In an effort to improve the activity of the reporter enzyme in neutral pH ranges, the gusA gene was cloned into the broad host range vector, pGK12, and subjected to random mutagenesis by passage through Epicurian coli mutator strain XL1-Red. Two mutant alleles, gusA2 and gusA3, were recovered that produced beta-glucuronidase with increased activity in neutral pH ranges. One of these, gusA3, was significantly more active in the pH range of 4-8 in both Escherichia coli and L. gasseri. Sequence analysis of gusA2 and gusA3 revealed single base pair changes that resulted in D524G and D573A substitutions, respectively. The modified GusA3 enzyme has expanded potential for use as a reporter enzyme in expression hosts that are not acidophilic, as well as lactic acid bacteria and other microorganisms that grow in acidifying environments.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

A new repressible alkaline phosphatase inNeurospora crassa EM 5297a

Neurospora crassa Em 5297a can utilize sodium Β-glycerophosphate as a sole phosphorous source (in the place of KH2PO4). Under these conditions a repressible alkaline phosphatase is elaborated which has different pH optimum towards Β-glycerophosphate (10.2) and pyrophosphate (9.0) as substrates. This enzyme does not require any metal ion for its activity and could be assayed in the presence of EDTA. However, under conditions of cobalt toxicity, the activity of this enzyme is high and is decreased in copper and nickel toxicities.     

Cloning of the DNA repair genes mtcA, mtcB, uvsC, uvsD, uvsE and the leuB gene from Deinococcus radiodurans

A gene library from Deinococcus radiodurans has been constructed in the cosmid pJBFH. A 51.5-kb hybrid cosmid, pUE40, that transduced Escherichia coli HB101 from leucine dependence to independence was selected, and a 6.9-kb fragment which carried the leuB gene from D. radiodurans was subcloned into the EcoRI site of pAT153. The DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE, which code for two D. radiodurans UV endonucleases were identified by transforming appropriate repair-deficient mutants of D. radiodurans to repair proficiency with DNA derived from the gene library. Hybrid cosmid pUE50 (37.9 kb) containing an insert carrying both the mtcA and mtcB genes was selected and 5.6- and 2.7-kb DNA fragments carrying mtcA and mtcB, respectively, i.e., the genes that code for UV endonuclease alpha, were subcloned into the EcoRI site of pAT153. The three genes uvsC, uvsD and uvsE, that code for UV endonuclease beta, were all present in the 46.0-kb hybrid cosmid pUE60. The uvsE gene in a 12.2-kb fragment was subcloned into the HindIII site of pAT153 and the size of the insert reduced to 6.1 kb by deletion of a 6.7-kb fragment from the hybrid plasmid pUE62. None of the uvs genes introduced into the UV-sensitive E. coli CSR603 (uvrA-) was able to complement its repair defect. The mtcA, uvsC, uvsD and uvsE genes were found in the 52.5-kb hybrid cosmid pUE70. It is concluded that the DNA repair genes mtcA, mtcB, uvsC, uvsD and uvsE are located within an 83.0-kb fragment of the D. radiodurans genome.     

Evolution of the RNA-dependent RNA polymerase (RdRP) genes: Duplications and possible losses before and after the divergence of major eukaryotic groups

Eukaryotic RNA-dependent RNA polymerases (RdRPs, encoded by RDR genes) play critical roles in developmental regulation, maintenance of genome integrity, and defense against foreign nucleic acids. However, the phylogenetic relationship of RDRs remains unclear. From available genome sequences, we identified 161 putative RDR genes from 56 eukaryotes, ranging from protists to multicellular organisms, including plants, fungi and invertebrate animals, such as nematodes, lancelet and sea anemone. On the other hand, we did not detect RDR homologs in vertebrates and insects, even though RNA interference functions in these organisms. Our phylogenetic analysis of the RDR genes suggests that the eukaryotic ancestor might have had three copies, i.e. RDRα, RDRβ and RDRγ. These three ancient copies were also supported by the patterns of protein sequence motifs. Further duplication events after the divergence of major eukaryotic groups were supported by the phylogenetic analyses, including some that likely occurred before the separation of subgroups within each kingdom. We present a model for a possible evolutionary history of RDR genes in eukaryotes.     

重组泡球蚴Em18蛋白不同纯化方法的比较

目的：比较不同方法纯化重组泡球蚴Em18的效果。方法：进一步对BL21-pET41a-Em18重组菌的诱导表达条件进行摸索优化,采用改良的超声程序破碎大肠杆菌细胞,分别经不同的纯化方法进行蛋白纯化,通过SDS-PAGE对纯化结果进行比较分析,Western blot进行活性鉴定。结果：①在菌液OD600为0.8-1.0,加IPTG终浓度为1 mmol/L,37℃诱导3h,rEm18-GST重组蛋白得到成功高表达,在相对分子量为50kDa处有表达条带。②超声程序为：工作3 s,间歇4s,功率200~300W,超声体系中加入溶菌酶和蛋白酶抑制剂作用,可促进大肠杆菌细胞的破碎,降低目的蛋白的降解;加入终浓度为1%的Triton-X100作用可增加融合蛋白的可溶性。③通过比较不同方法纯化重组泡球蚴Em18的效果表明采用单纯His柱纯化可获得浓度高、纯度高的rEm18-GST重组蛋白。Western blot分析表明该蛋白能与泡型包虫病（AE）患者血清特异性反应。结论：建立了一种纯化原核表达Em18融合蛋白的较为经济和有效的方法,得到大量有生物学活性的Em18融合蛋白,为包虫病诊断试剂盒的研制奠定基础。