香菇三个功能基因部分序列的单核苷酸多态性分析

以15个香菇栽培品种为材料,对尿嘧啶核苷酸-胞嘧啶核苷酸激酶基因(UMP-CMP kinase gene,uck1)、分裂原活化蛋白激酶基因(mitogen-activated protein kinase gene,mapk)和外切β-1,3葡聚糖酶基因(exo-β-1,3-glucanase-encoding gene,exg1)进行了部分序列的单核苷酸多态性(single nucleotide polymorphism,SNP)分析。结果表明,测序中出现的双峰,是菌丝双核体细胞中两细胞核之间的差异造成的。在采用uck1、mapk和exg1的3,126bp中,共发现48处多态性位点,发生频率为1/65bp,其中36个属于转换、12个为颠换。从群体发生频率上,38个属于超过10%的常见SNP,10个属于罕见SNP。不同基因的SNP发生频率不同,uck1、mapk和exg1的SNP发生频率分别为1.40%、0.82%和2.41%。外显子区SNP数量高于内含子,3个基因在外显子区域分布28个,内含子分布20个。外显子的28个SNP位点中,11个为错义突变,17个为同义突变。错义突变引起了编码氨基酸的改变。对SNP位点的聚类分析表明,15个栽培品种间存在的多态性位点在1–36之间,15个品种的SNP类型不同。uck1,mapk,exg1的SNP可用于香菇栽培品种的鉴别。     

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) from the mushroom Lentinula edodes: characterization of the cDNA and its expressed product

A cDNA homologue of Schizosaccharomyces pombe cdc5(+) was isolated from the basidiomycete mushroom Lentinula edodes and it was named Le.cdc5 cDNA. The deduced Le.CDC5 (842 amino acid residues) possessed N-terminal amino acid sequence highly homologous to those of S. pombe cdc5(+) gene product (Sp.cdc5p) and Sp.cdc5p-related proteins (SPCDC5RPs). The N-terminal 185 amino acid peptide of Le.CDC5 (Le.CDC5(1-185) peptide) produced in Escherichia coli was subjected to random binding-site selection analysis, revealing that Le.CDC5(1-185) peptide binds to a 7-bp sequence with the consensus sequence of 5'GCAATGT3' (complementary; 5'ACATTGC3'). Genomic binding-site (GBS) cloning by using Le.CDC5(1-185) peptide resulted in an isolation of the DNA fragment that contained three sets of 7-bp consensus-like sequence and TATA box. The Le.CDC5 protein contained two putative phosphorylation sites of cAMP-dependent protein kinase (A kinase) in its C-terminus. There exists a possible leucine zipper between the two phosphorylation sites. The Le.CDC5 fragment containing the two phosphorylation sites was actually phosphorylated by commercially available A kinase. Yeast two-hybrid analysis suggested the homodimerization of Le.CDC5 protein probably through the leucine zipper. Northern blot analysis showed that Le.cdc5 gene is most actively transcribed in primordia and small immature fruiting bodies of L. edodes, implying that Le.cdc5 may play a role in the beginning and early stage of fruiting-body formation.     

cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli

A cDNA coding for UMP-CMP kinase from Dictyostelium discoideum was isolated from a lambda gt11 expression library and sequenced. The corresponding mRNA has a size of 0.7 kilobase and is down-regulated during early development of D. discoideum. Southern blotting demonstrated that the UMP-CMP kinase is encoded by a single gene. The deduced amino acid sequence of UMP-CMP kinase shows a high degree of homology with adenylate kinases from different sources with the highest degree of homology to cytosolic adenylate kinase from vertebrate muscle (43%). The enzyme expressed in Escherichia coli after cloning the cDNA into an ATG expression vector was purified and analyzed for its structural and kinetic properties. The UMP-CMP kinase uses preferentially ATP (Km,app = 25 microM) as phosphate donor and is specific for UMP (Km,app = 0.4 mM) and CMP (Km,app = 0.1 mM). The enzyme is strongly inhibited by the substrate analogue P1-(adenosine-5')-P5-(uridine-5')-pentaphosphate (Ki between 0.05 and 0.1 microM) and is inactivated by modification of free thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid).     

Novel odorant-binding proteins expressed in the taste tissue of the fly

A taste tissue cDNA library of the fleshfly Boettcherisca peregrina was screened with a subtracted cDNA probe enriched with taste-receptor-tissue-specific cDNA. Seven genes were identified with sequence similarity to insect odorant-binding protein (OBP) genes. The predicted amino acid sequences of the genes contain the putative signal peptide sequence at the N-terminal and most of them conserve the six cysteines common to known insect OBPs. These genes show a high degree of sequence divergence with approximately 20% amino acid identity. The most striking feature was that all seven of these genes are expressed mainly in the taste tissues, such as the labellum and tarsus, unlike the known insect OBP genes expressed in olfactory tissue. The predicted amino acid sequences had the highest degree of sequence similarity to the Drosophila melanogaster OBPs named pheromone binding protein-related proteins (PBPRPs). These gene products are here referred to as gustatory PBP-related proteins (GPBPRPs) 1-7. Homologous GPBPRP genes were found also in D. melanogaster by database search and are shown to be expressed in Drosophila taste tissues.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

cGMP-dependent protein kinase genes in Drosophila

Two Drosophila genes encoding products related to cGMP-dependent protein kinase have been isolated by cross-hybridization to a Drosophila cAMP-dependent protein kinase catalytic subunit gene. Both genes encode products with putative cGMP binding and kinase domains on the same polypeptide chain, as found for the prototypical bovine lung cGMP-dependent protein kinase. The deduced product of one gene (DG1; cytological position, 21D) is 14% larger than the bovine enzyme and differs substantially in sequence at the amino terminus, the region responsible in the bovine enzyme for dimerization. The second gene (DG2; cytological position, 24A) is transcribed into three major RNA species of different size. The largest (DG2; T1) and smallest (DG2;T3) RNAs encode overlapping polypeptides of similar sequence to the whole length of bovine lung cGMP-dependent protein kinase. The translation product of the third major RNA (DG2;T2) lacks sequences similar to those that constitute the dimerization and kinase inhibitory domains of the bovine enzyme. The percentage amino acid identity between DG1 or DG2 and bovine lung cGMP-dependent protein kinase is 55 and 64%, respectively. A common progenitor of the two cGMP-dependent protein kinase genes, DG1 and DG2, is strongly suggested by the conserved positions of introns in these genes.     

In vitro binding to the leucine tRNA gene identifies a novel yeast homeobox gene

In a search for gene products of Saccharomyces cerevisiae interacting with the internal promoter of yeast tRNA genes two genes encoding a homeodomain protein of the Drosophila Antennapedia type were isolated. One of them codes for Pho2, and the second codes for a previously unknown protein (Yox1). The corresponding gene, termed YOX1, maps to chromosome 16. The amino acid sequence of Yox1 shows a remarkable similarity within the homeobox domain to many proteins from a wide variety of sources. Fusion proteins that contain sequences encoded by these genes demonstrate that the genes encode DNA-binding proteins that are capable of binding to the DNA of the leucine tRNA gene in vitro. However, deletion of YOX1 gene activity does not give rise to a scorable mutant phenotype. This result leaves open whether Yox1 binding to the leucine tRNA gene is necessary for the in vivo regulaiton of the gene and its suggests that the YOX1 gene codes for a nonessential product.by H. JäckleThe sequence data reported here will appear in the EMBL, Gen-Bank and DDBJ Nucleotide Sequence Databases under the accession number X62392     

Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases

The complete nucleotide sequence of a 2.9-kb DNA fragment containing the CDC2 gene-complementing activity from Schizosaccharomyces pombe has been determined. Within this region lies a 1.69-kb DNA sequence whose predicted amino acid sequence shows extensive homology to that previously deduced for the CDC28 gene product from Saccharomyces cerevisiae [L?rincz and Reed, Nature 307 (1984) 183-185]. Taken with the earlier observation that mutants in CDC2 can be rescued by the presence of the CDC28 gene [Beach, Durkacz and Nurse, Nature 300 (1982) 706-709], these results strongly suggest that the two genes code for similar functions. In contrast to the CDC28 gene, however, which contains no introns, the CDC2 coding sequence is split by four introns and from a comparison of the two sequences a consensus sequence for intron splicing in S. pombe can be established. Both CDC2 and CDC28 contain the consensus sequences for the ATP binding and phosphorylation acceptor sites of protein kinases such as bovine cAMP-dependent protein kinase (bov PK) and the src family of viral oncogene products.