Cloning and Characterization of the nagA Gene that Encodes β-N-Acetylglucosaminidase from Aspergillus nidulans and Its Expression in Aspergillus oryzae

We isolated a β-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed β-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of β-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture.     

Cloning of a Complementary DNA that Encodes an Acidic Chitinase which is Induced by Ethylene and Expression of the Corresponding Gene

A complementary DNA encoding an ethylene-inducible acidic chitinaseof azuki bean (Vigna angularis) was isolated, and its completenucleotide sequence was determined. The nucleotide and deducedamino-acid sequence were very similar to those of an acidicchitinase from cucumber leaves that had been infected with tobacconecrosis virus. The mRNA for the acidic chitinase was not detectedin leaves of azuki bean that had not been treated with ethylene,but it appeared 3 h after initiation of treatment with ethyleneand its level gradually increased over a period of 19 h. ThemRNA also accumulated in response to salicylate or wounding.The expression of the gene in response to wounding was suppressedby 2,5-norbornadiene, but that in response to salicylate wasnot affected by this inhibitor. (Received May 19, 1992; Accepted November 2, 1992)     

类大麦属高分子量谷蛋白基因Kx的序列测定及表达

利用SDS-PAGE检测了2份类大麦属(Crithopsis delileana)材料的高分子量谷蛋白亚基组成,并对其中1份材料的x型亚基进行了克隆和测序。结果表明,2份材料具有完全相同的蛋白电泳图谱。在小麦的高分子量区域仅检测到一条蛋白质带,与小麦y型亚基的迁移率接近,但克隆测序表明其为x型高分子量谷蛋白亚基,其编码基因命名为Kx。Kx基因编码区序列长度为2052bp．编码长度为661个氨基酸残基的蛋白质,其序列具有典型的x型高分子量谷蛋白亚基的特征。Kx基因能在原核表达系统内正确表达,其表达蛋白与来源于种子中的Kx亚基的迁移率完全一致。Kx亚基与小麦属A、B和D,山羊草属C和U以及黑麦属R染色体组编码的高分子量谷蛋白亚基氨基酸序列非常相似,但在N和C保守区的氨基酸组成以及重复区长度上与它们存在明显差异。聚类分析可将Kx与Ax1聚类为平行的分支。由此可见,来源于C．delileana的Kx基因为一新的x型高分子量谷蛋白亚基基因。     

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς²⁸-like and ς⁷⁰-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.     

黄瓜扩张蛋白基因CsEXP10的克隆与表达

以cDNA-AFLP差示片段的序列（CO434610）为基础,通过RACE延伸和与EST序列拼接,得到长度为1191bp的、包含完整3’末端的CsEXP10基因cDNA序列。Southern杂交结果表明,该基因在黄瓜基因组中以单拷贝形式存在．RT-PCR检测发现,该基因不在根、茎和叶中表达,而在果实中表达．Northern杂交显示,该基因在授粉后迅速生长的幼果中丰量表达,而在幼小子房、开花当天的未授粉子房和生长停止的果实中不表达,由此推测CsEXP10基因与授粉后黄瓜果实膨大生长有密切关系。     

Evidence for an Association of the 15-kDa Proteolipid of Mediatophore with a 14-kDa Polypeptide

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetylcholine on calcium action, forms a complex with a 14-kDa polypeptide. The complex was identified based on the following results. (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15- and 14-kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak. (c) After 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti-14-kDa antigen monoclonal antibody retained both the 14-kDa and the 15-kDa polypeptide. Similarly, immunoprecipitation experiments using protein A-coated beads sedimented an immunocomplex in which both antigens were found. (d) The 14-kDa antigen could be localized in the synaptosomal membrane where mediatophore and its 15-kDa component are found.     

地衣芽孢杆菌2709碱性蛋白酶基因的克隆、序列测定及其表达

以克隆的地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增片段为探针。通过原位杂交从2709基因文库中筛选出两个含有完整的2709碱性蛋白酶基因的阳性克隆：Psci和Psc7。对Psc7中的插入片段构建若干亚克隆后测定了其全部DNA序列,结果显示该插入片段含2709碱性蛋白酶及其信号肽与导肽(Pro—peptide)在内的全部编码序列(1140碱基对)及长度分别为299和832碱基对的上、下游序列,该序列同M．Jacobs等克隆的地衣芽孢杆菌NcIB 6816的subtlisin Carlsberg基因序列显示了极高的同源性。通过枯草杆菌-大肠杆菌穿梭质粒Pbe2将克隆的2709碱性蛋白酶基因转入到蛋白酶缺陷型的枯草芽孢杆菌DB104中,结果表明2709碱性蛋白酶基因在枯草芽孢杆菌中得到了明显的表达。     

The Chlamydomonas IDA7 Locus Encodes a 140-kDa Dynein Intermediate Chain Required to Assemble the I1 Inner Arm Complex

To identify new loci that are involved in the assembly and targeting of dynein complexes, we have screened a collection of motility mutants that were generated by insertional mutagenesis. One such mutant, 5B10, lacks the inner arm isoform known as the I1 complex. This isoform is located proximal to the first radial spoke in each 96-nm axoneme repeat and is an important target for the regulation of flagellar motility. Complementation tests reveal that 5B10 represents a new I1 locus, IDA7. Biochemical analyses confirm that ida7 axonemes lack at least five I1 complex subunits. Southern blots probed with a clone containing the gene encoding the 140-kDa intermediate chain (IC) indicate that the ida7 mutation is the result of plasmid insertion into the IC140 gene. Transformation with a wild-type copy of the IC140 gene completely rescues the mutant defects. Surprisingly, transformation with a construct of the IC140 gene lacking the first four exons of the coding sequence also rescues the mutant phenotype. These studies indicate that IC140 is essential for assembly of the I1 complex, but unlike other dynein ICs, the N-terminal region is not critical for its activity.     

Cloning and Characterization of the nrtA Gene That Encodes a 45-kDa Protein Involved in Nitrate Transport in the Cyanobacterium Synechococcus PCC 7942

A 45-kDa protein in the cytoplasmic membrane of the cyanobacteriumSynechococcus PCC 7942 is involved in the active transport ofnitrate [Omata et al. (1989) Proc. Natl. Acad. Sci. USA 86:6612]. The gene coding for this protein (designated herein asnrtA) has been cloned and sequenced. The nrtA gene encodes aprotein of 443 amino acids with a calculated molecular weightof 48424. The deduced amino acid sequence of the protein is46.5% homologous to that of a 42-kDa cytoplasmic membrane proteinthat is synthesized under carbon-limited conditions in SynechococcusPCC 7942. (Received July 16, 1990; Accepted December 5, 1990)     

Nucleotide Sequence of the Gene That Encodes a Neopullulanase from an Alkalophilic Bacillus

A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40. The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments. The enzyme was identified as a 31 kDa protein which is probably an N-acetylmuramyl-L-alanine amidase. The amino-terminal 15 residues of the enzyme were: VLPEPVVPEYIVVHN.     

Relation between Sex Expression Types and Cotyledon Etiolation of Cucumber in vitro I. On the Role of Ethylene Evolved from Seedlings

Cucumber seedlings, when cultured in vitro, showed differencesin cotyledon etiolation rates among cultivars with differentgenetic backgrounds for sex expression. The chlorophyll contentin gynoecious cultivars (acr^F/acr^F) decreased rapidly whilethat in monoecious ones (acr⁺/acr⁺) decreased more slowly, andthat in mono-gynoecious ones (acr¹/acr¹) decreased at an intermediaterate. Etiolation was suppressed even in early-etiolating cultivarswhen the flask remained unsealed or endogenously evolved ethylenewas removed. Cotyledon etiolation was enhanced even in late-etiolatingcultivars when ethephon was added to the flask. The rate ofetiolation corresponded to the ethylene concentration in theflask; much more ethylene was detected in early-etiolating cultivarsthan in late-etiolating ones. Ethylene accumulation is one of the important factors involvedin the cotyledon etiolation observed in in vitro cultures. Thedifference in etiolation rates among seedlings with differentgenetic backgrounds for sex expression corresponds to theirability for ethylene evolution, in the order of acr^F>acr¹>acr⁺. (Received January 6, 1981; Accepted March 23, 1981)