The correlation of the gene csoS2 of the carboxysome operon with two polypeptides of the carboxysome in Thiobacillus neapolitanus

The carboxysomal polypeptides of Thiobacillus neapolitanus with apparent molecular masses of 85 and 130 kDa were isolated and subjected to N-terminal sequencing. The first 17 amino acids of the two peptides were identical. The sequence perfectly matched the deduced amino acid sequence of an open reading frame in the carboxysome operon. The gene was subsequently named csoS2. Expression of the gene in Escherichia coli resulted in the production of two peptides with apparent molecular masses of 85 and 130 kDa. Immunospecific antibodies generated against the smaller peptide recognized both peptides; the peptides were named CsoS2A and CsoS2B, respectively. A digoxigenin-hydrazide glycosylation assay revealed that both CsoS2A and CsoS2B are post-translationally modified by glycosylation. CsoS2 was localized to the edges of purified carboxysomes by immunogold electron microscopy using the monospecific CsoS2A antibodies. The molecular mass of CsoS2A calculated from the nucleotide sequence was 92.3 kDa. Received: 1 March 1999 / Accepted: 29 June 1999     

Identification and localization of the carboxysome peptide Csos3 and its corresponding gene in Thiobacillus neapolitanus

Four genes encoding carboxysome shell peptides (csoS1A, csoS1B, csoS1C, csoS2), the genes encoding the large and small subunits of RuBisCO (cbbL, cbbS), and three unidentified ORFs constitute an operon in Thiobacillus neapolitanus. An unidentified ORF 1.54 kb in size is predicted from sequence analysis to encode a protein with a molecular mass of approximately 57 kDa. When this ORF was expressed in Escherichia coli under the control of its endogenous ribosome-binding site, no peptide product was observed. In order to correlate this ORF with a carboxysome peptide, the ORF was overexpressed in E. coli by cloning it into pProExHTb, a prokaryotic expression vector containing an E. coli ribosome binding site. When antibodies raised against the recombinant protein were used to probe an immunoblot containing carboxysome peptides, a 60-kDa peptide was recognized. The peptide was subsequently named CsoS3. CsoS3 is a minor component of the carboxysome; a peptide of this size is commonly not observed or is very faint on Coomassie blue-stained SDS-polyacrylamide gels of purified carboxysomes. Immunogold labeling established CsoS3 to be a component of the carboxysome shell.     

Using bacteria to analyze sequences involved in chloroplast gene expression

The complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. Other genes involved in photosynthesis, respiratory electron transport, and membrane-associated transport in chloroplasts were predicted by the amino acid sequence homology and secondary structure of gene products. Thirty-three open reading frames in the liverwort chloroplast genome remain unidentified. However, most of these open reading frames are also conserved in the chloroplast genomes of two species, a liverwort, Marchantia polymorpha, and tobacco, Nicotiana tabacum, indicating their active functions in chloroplasts.Abbreviations bp base pair - kDa kilodalton - IR inverted repeat - ORF open reading frame - DALA -aminolevulinate     

棉花抗逆基因GhAREB4的克隆及功能分析

AREBs转录因子家族基因主要参与干旱、高盐、低温等胁迫应答反应,在植物抵御各种逆境胁迫中起着非常重要的作用。该研究经序列电子拼接克隆了陆地棉GhAREB4基因,该基因全长1 784bp,其开放阅读框为1 227bp,编码408个氨基酸,预测分子量为44.3kD,等电点为8.88。蛋白结构预测发现,该蛋白二级结构中含有bZIP基因家族的保守结构域。系统进化树分析表明,GhAREB4与可可的AREB转录因子同源性最高。绿色荧光蛋白亚细胞定位分析表明,GhAREB4蛋白分布在细胞核内。qRT-PCR分析表明,GhAREB4基因在花中的表达量最高;且GhAREB4基因表达受到干旱、高盐、低温、脱落酸(ABA)等处理的诱导,其可能调控棉花对非生物逆境的耐性响应。研究结果为进一步研究该基因对棉花耐逆调控机制奠定了基础。     

IS1237, a Repetitive Chromosomal Element from Clavibacter xyli subsp. cynodontis, Is Related to Insertion Sequences from Gram-Negative and Gram-Positive Bacteria

We describe here a repetitive chromosomal element, which appears to be an insertion sequence, isolated from Clavibacter xyli subsp. cynodontis, a gram-positive plant-associated bacterium. The element, IS1237, is 905 bp in size, is bounded by 19-bp perfect inverted repeats and 3-bp direct repeats, and appears at least 16 times in the genome. It contains three open reading frames which show similarity to open reading frames from various other insertion sequences. We have found that there are two groups of related mobile elements: one in which two open reading frames are read separately and the other in which these two open reading frames are fuse together to give one predicted protein product. Using one of these open reading frames to search amino acid sequence databases, we found two instances in which similar reading frames flank genes carried on plasmids. We believe therefore that these plasmid-borne genes may be parts of previously unidentified mobile elements. For IS1237, a frameshift in two of the open reading frames and a stop codon in the third may indicate that this particular copy of the element is no longer active in transposition. The similarity of IS1237 to other elements from both gram-negative and gram-positive bacteria provides further evidence that mobile elements have been transferred between these two bacterial groups.     

长春花丙二烯氧化物合酶基因的克隆与表达

利用RT-PCR和RACE相结合的方法,从长春花中克隆了丙二烯氧化物合酶(AOS)基因。结果显示:长春花AOS基因(CrAOS)cDNA全长为2 118bp,包括5′和3′非翻译区,polyA尾和一个长1 638bp的开放阅读框,其基因组中不含内含子;CrAOS基因编码的蛋白含545个氨基酸。多重比对表明CrAOS蛋白与其他的AOS蛋白具有较高的相似性,CrAOS蛋白序列中含有AOS家族应有的保守氨基酸残基。Southern杂交表明:CrAOS基因在长春花中为低拷贝。qRT-PCR结果显示:CrAOS在各个组织均有表达但表达量存在差异,在老叶中最高,在幼花中表达最低。对长春花幼苗进行不同处理,结果表明:伤害、低温、甲基茉莉酸、乙烯利处理等可使CrAOS基因表达量显著提高,水杨酸处理对基因表达影响不大。     

Analysis of the Complete Nucleotide Sequence of the Tetracycline-Resistance Transposon Tn10

An analysis of the complete nucleotide sequence of the composite tetracycline-resistance transposon Tn10 (9147 bp) from the Salmonella typhi conjugative plasmid R27 is presented. A comparison of the protein sequences from IS10-right and IS10-left transposases has identified four amino acid differences. These residues appear to play an important role in normal transposase function and may account for the differences in exhibited transposition activities. The tetracycline determinants encoded by this version of Tn10 share >99% identity with those of Tn10^R100, demonstrating the conservation that exists between these transposons. A previously uncharacterized 3000-bp region of Tn10 contains four putative open reading frames. One of these open reading frames shares 55% identity with the glutamate permease protein sequence from Haemophilus influenzae although it was unable to complement an Escherichia coli glutamate permease mutant, with which it shares 51% identity. The three remaining putative open reading frames are arranged as a discrete genetic unit adjacent to the glutamate permease homolog and are transcribed in the opposite direction. Two of these open reading frames are homologous with Bacillus subtilis proteins of unknown functions while the other has no homologs in the database. The presence of an aminoacyl-tRNA synthetase class II motif in one of these open reading frames in combination with the glutamate permease homolog allows us to postulate that this region of Tn10 could once have played a role in amino acid metabolism.     

小立碗藓PpAGO7基因的克隆及表达分析

该研究采用同源克隆的方法获得了小立碗藓AGO7蛋白编码基因PpAGO7,对PpAGO7基因序列特征进行生物信息学分析,并利用Real-time PCR方法分析PpAGO7基因的时空表达模式,为揭示小立碗藓中PpAGO7基因的生物学功能提供依据。结果表明:PpAGO7基因的全长cNDA为3 583bp,其中开放阅读框3 363bp,编码1 120个氨基酸,分子量123.38kD,等电点9.26,含有AGO蛋白典型的PAZ和PIWI结构域;PpAGO7基因在小立碗藓整个生活周期都有表达,且在茎叶体生长时期表达水平较高,但在原丝体时期表达水平较低。研究结果推测PpAGO7基因可能在茎叶体拟叶的生长发育过程中起作用。     

Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from <Emphasis Type="Italic">Aspergillus usamii</Emphasis> E001

The full-length cDNA gene that encodes an acidophilic endo-1,4-β- xylanase XynI of Aspergillus usamii E001 was amplified by rapid amplification of cDNA 3′ and 5′ ends (RACE) using the total RNA as template and then cloned onto the pUCm-T vector, followed by sequencing. The cloned cDNA is 881 bp in length including 5′ and 3′ non-encoding regions, as well as a 678 bp of open reading frame (ORF) which encodes an E001 XynI of 188 amino acid residues together with a signal peptide of 37 amino acid residues. The homologies of E001 XynI with xylanases of Aspergillus niger, Aspergillus kawachii, Emericella nidulans and Penicillium funiculosum are 97.8, 92.0, 74.6 and 60.5%, respectively. From a BLAST search result, we concluded that E001 XynI belongs to the glycoside hydrolase family 11. Its three-dimensional structure was predicted using programs based on that of the P. funiculosum xylanase (1TE1B) from the family 11. In addition, the complete DNA gene xynI encoding E001 XynI was cloned from the genomic DNA of A. usamii E001 by conventional PCR and ligation-mediated PCR amplification. The cloned xynI is 1,206 bp in length, composed of a promoter region, a 68 bp of intron and two exons when compared with the cDNA of E001 XynI.     

烟草氧化胁迫相关基因NtOSA1的克隆表达及亚细胞定位分析

植物类蛋白激酶Abc1家族(activity of bc1complex)在植物生长发育及响应非生物胁迫中起着重要的作用。该研究通过将拟南芥AtOSA1蛋白氨基酸序列与烟草转录组数据进行比对,利用巢式PCR技术克隆得到1个烟草氧化胁迫相关Abc1家族基因NtOSA1,NtOSA1与拟南芥AtOSA1基因具有72.89%的一致性。NtOSA1基因开放阅读框长度为2 283bp,编码760个氨基酸,含有典型的ABC1结构域、一个激酶结构域、叶绿体定位信号肽和两个跨膜结构域。采用实时荧光定量PCR技术,对NtOSA1基因在烟草不同组织以及氧化胁迫、盐胁迫等处理下的表达分析表明:NtOSA1基因的表达具有组织特异性,主要在叶片中表达;NtOSA1基因在H2O2和NaCl处理后表达量上升,均在处理后6h达到最大值,分别为处理前的1.95和2.69倍。亚细胞定位结果表明,NtOSA1蛋白定位在细胞叶绿体上,与预测结果一致。研究表明,NtOSA1基因参与了烟草抗氧化胁迫和盐胁迫的响应。     

Cloning and Nucleotide Sequence of the Endoinulinase-Encoding Gene, inu2, from Aspergillus ficuum

A 2.3 kb DNA fragment that contains a gene encoding endoinulinase, inu2, from Aspergillus ficuum ATCC 16882 was isolated and analyzed. It includes an open reading frame of 1,551 bp, coding for a polypeptide with calculated molecular weight of 55,790 Da, including a putative signal peptide of 22 amino acids. Alignment of amino acid sequences revealed 73.3% identity and 93.9% similarity between A. ficuum and Penicillium purpurogenum endoinulinase.     

割手密醛脱氢酶ScALDH基因的克隆与表达分析

该研究从甘蔗细茎野生种(割手密Saccharum spontaneum)中克隆抗旱相关的基因Sc ALDH,并分析其在干旱处理条件下的表达情况和序列特征。利用RT-PCR技术克隆甘蔗的ALDH基因片段,并对其核苷酸和氨基酸序列进行分析。使用NCBI Blastx、ORF finder、Mega、NCBI Conserved Domain Search等程序对其分别进行不同物种氨基酸比较、开放阅读框(ORF)寻找、进化树及保守序列分析,并用Real Time-PCR分析所克隆基因在干旱胁迫前后的表达差异。结果表明:克隆出甘蔗的ALDH基因片段,总长度为1996bp,其中蛋白质编码区(CDS)全长1524bp,编码508个氨基酸;与其它物种ALDH类蛋白氨基酸序列有很高的同源性,有ALDH家族的保守序列,并含有完整的开放阅读框,系统进化树分析显示与玉米的蛋白质亲缘关系最近;Real timePCR数据表明,在干旱胁迫下,随着干旱时间的延长,该基因的表达量呈持续积累的表达模式。总体上来说,该基因对干旱胁迫显著表达。利用RT-PCR技术克隆的甘蔗ALDH基因,属于ALDH蛋白家族的一员,具有其典型的功能域,该基因在干旱胁迫过程中参与抗旱作用。该研究结果为野生种资源开发、优良抗旱亲本选择和培育抗旱性强甘蔗品种提供了参考依据。     

Cloning and Nucleotide Sequence of the Gene Encoding a Serine Proteinase Inhibitor Named Marinostatin from a Marine Bacterium,Alteromonas sp. Strain B-10-31

The gene (mstI) encoding a serine proteinase inhibitor named marinostatin from marine Alteromonas sp. strain B-10-31 was cloned and its nucleotide sequence was analyzed. A short open reading frame of 192 bp encoded 63 amino acids with a molecular weight of 6,985. Furthermore, the initial product of marinostatin (marinostatin L) was purified and its amino acid sequence was analyzed. These results indicate that marinostatin is produced as a unique precursor consisting of the mature peptide and the leader peptide for an ATP-binding cassette (ABC) transporter, and furthermore the initial product of marinostatin is dehydrated and processed by proteolysis to give homologous forms of marinostatin.     

DNA sequence and genome organization of genital human papillomavirus type 6b.

The complete nucleotide sequence of the circular double-stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1). All major open reading frames are located on one DNA strand only. Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1. The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames. The internal part of E2 completely overlaps with another open reading frame E4. The putative late region contains two large open reading frames L1 and L2. The L1 amino acid sequences are highly conserved among analyzed papillomavirus types. By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression.     

Identification of two novel xylanase-encoding genes (xyn5 and xyn6) from Acrophialophora nainiana and heterologous expression of xyn6 in Trichoderma reesei

Two novel genes, xyn5 and xyn6, coding for family 11 xylanases, were isolated from the thermotolerant filamentous fungus, Acrophialophora nainiana, by PCR using degenerate primers. The xyn6 gene was further expressed in Trichoderma reesei. DNA sequence analysis of xyn6 revealed an open reading frame (ORF) of 708 bp, interrupted by an intron of 58 bp. The xyn6 ORF encodes a predicted protein of 236 amino acid residues. The mature recombinant XynVI protein had a molecular mass of about 19 kDa, as estimated by gel electrophoresis. Analysis of the predicted amino acid sequence of XynVI paves the way for rational protein engineering by site-directed mutagenesis aiming to increase the thermostability of the heterologously-expressed xylanase.     

Peroxyl Radical Scavenging Activity of Caffeic Acid and Its Related Phenolic Compounds in Solution

The esterase gene (est) of Pseudomonas putida MR-2068 was cloned into Escherichia coli JM109. An 8-kb inserted DNA directed synthesis of an esterase in E. coli. The esterase gene was in a 1.1-kb PstI-ClaI fragment within the insert DNA. The complete nucleotides of the DNA fragment containing the esterase gene were sequenced and found to include a single open reading frame of 828 bp coding for a protein of 276 amino acid residues. The open reading frame was confirmed by N-terminal amino acid sequence analysis of the purified esterase. A potential Shine-Dalgarno sequence is followed by the open reading frame. The esterase activity of the recombinant E. coli was more than 200 times higher than that of parental strain, P. putida MR-2068.     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

Characterization,expression, and functional analysis of testis-specific serine/threonine kinase 1 (Tssk1) in the pen shell Atrina pectinata

Abstract

Testis-specific serine/threonine kinase 1 (Tssk1) was named since its function was determined in mouse spermiogenesis. In our research, we cloned a homolog of the pen shell Atrina pectinata Tssk1, ApTssk1, and determined its expression characteristics at mRNA. The full length of ApTssk1 cDNA was 1517 bp, with an open reading frame of 1089 bp (58–1146), which encodes a peptide including 362 amino acids. The homologous analysis indicated that the deduced amino acid sequences of ApTssk1 shared a close identity with other reported Tssk1. In addition, the highly conserved fragment containing a serine/threonine protein kinase catalytic (S-TKc) domain was predicted to exist in ApTssk1. Results from the RT-qPCR display that ApTssk1 was only transcribed in the male gonad of A. pectinata, and was highest in the mature testis. Therefore, this study indicated that ApTssk1 might play a functional role during spermatogenesis in A. pectinata.     

丹参多酚氧化酶基因的克隆及表达分析

前期研究发现多酚氧化酶(PPO)能正向调控丹酚酸B合成,该研究运用RACE技术,从丹参毛状根中克隆到多酚氧化酶基因(SmPPO,GenBank登录号为KF712274)全长序列,其cDNA全长1 930bp,开放阅读框为1 770bp,编码589个氨基酸。将SmPPO与管状花目其它4个物种进行氨基酸序列比对,在N端类囊体转移结构域中发现都存在2个N-豆蔻酰化位点。在丹参毛状根培养液中加入不同诱导因子,利用实时荧光定量PCR检测,发现该基因在酵母提取物处理中表达量显著上调,但在银离子、抗坏血酸和L-半胱氨酸处理中表达受到明显抑制。运用HPLC技术同步检测毛状根中丹酚酸B含量,显示出与基因表达相同的变化趋势。研究表明,丹参中多酚氧化酶基因(SmPPO)对丹酚酸B的合成具有正向调控作用。