Cloning and expression analysis ofMBLL cDNA

Thembl (muscleblind) gene ofDrosophila encodes a nuclear protein which contains two Cys₃His motifs. The mutation ofmbl gene will disturb the differentiation of all theDrosophila’s photoreceptors. Primers have been designed according to human EST086139, which is highly homologous tombl gene. Human fetal brain cDNA library has been screened and a novel cDNA clone has been obtained. The 2595 bp cDNA, designatedMBLL (muscleblind-like), contains an open reading frame which encodes 255 amino acids and has 4 Cys₃His motifs (GenBank Acc. AF061261). The amino acids sequence shares high homology toDrosophila’s mbl. The Northern blot and RNA dot blot hybridization of 43 human adult tissues and 7 fetal tissues show thatMBLL is a widely expressed gene, but the expression amounts differ in these tissues.     

A categorical interpretation of Yetter-Drinfel’d modules

It is known that any strict tensor category (C,⊗,I) can determine a strict braided tensor categoryZ(C), the centre ofC. WhenA is a finite Hopf algebra, Drinfel’d has proved thatZ( _AM) is equivalent to_D(A)M as a braided tensor category, where_AM is the left A-module category, andD(A) is the Drinfel’d double ofA. This is the categorical interpretation ofD(A). Z( _AM) is proved to be equivalent to the Yetter-Drinfel’d module category,_AYD ^A as a braided tensor category for any Hopf algebraA. Furthermore, for right A-comodule categoryM ^A, Z(M^A) is proved to be equivalent to the Yetter-Drinfel’d module category_AY D^A as a braided tensor category. But,in the two cases, the Yetter-Drinfel’d module category_AY D^A has different braided tensor structures.     

Fine mapping of the rice bacterial blight resistance gene Xa-4 and its co-segregation marker

An F₂ population developed from theXa-4 near isogenic lines, IR24 and IRBB4, was used for fine mapping of the rice bacterial blight resistance gene,Xa-4. Some restriction fragment length polymorphism (RFLP) markers on the high-density map constructed by Harushima et al. and the amplified DNA fragments homologous to the conserved domains of plant disease resistance (R) genes were used to construct the genetic linkage map around the geneXa-4 by scoring susceptible individuals in the population.Xa-4 was mapped between the RFLP marker G181 and the polymerase chain reaction (PCR) marker M55. The R gene homologous fragment marker RS13 was found co-segregating withXa-4 by analyzing all the plants in the population. This result opened an approach to map-based cloning of this gene, and marker RS13 can be applied to molecular marker-assisted selection ofXa-4 in rice breeding programs.     

Efficacy of anti-CD20 chimeric Fab’ fragment on proliferation of B lymphoma cells

The variable domain of heavy chain (V_H) and light chain (V_L) genes of anti-CD20 monoclonal antibody HI⁴⁷ were cloned from anti-CD20 ScFv expression vector pCANBTEcd20 by PCR and ligated into vector pYZF to construct chimeric anti-CD20 Fab’ fragment expression vector pYZFcd20. Chimeric anti-CD20 Fab’ fragment was expressed inE. coli 16C9 and purified by protein G affinity chromatography. Competitive inhibition assay showed that anti-CD20 Fab’ fragment inhibited binding of HI⁴⁷ to CD20 on the surface of Daudi cells. Results from MTT assay indicated that chimeric anti-CD20 Fab’ fragment inhibited the proliferation of Daudi cells, IC₅₀ = 69 μg/mL. Affinity of chimeric anti-CD20 Fab’ fragment was determined, K_a was about 8.9×10⁸ (mol/L)⁻¹.     

Cloning, sequencing and expression of a swine IgG H chain gene inE. coli

A DNA fragment about 1.5 kb has been isolated from spleen of adult Chinese swine by RT-PCR. The DNA fragment encodes immunoglobulin IgG H chain gene. Sequencing analysis showed that the DNA fragment is 1 425 bp long, complete CDS. The C region of the gene has been classified as Subclass Ig γ3, and is the same as reported by Sun et al., but V region of the present gene is 42 bp less by comparison. The gene has been ligated into expression vector pET-3b (NSEB)( - ). A protein about 52 ku has been expressed in E. coli with an expression level of about 21 % .     

GCC box in<Emphasis Type="Italic">Arabidopsis PDF1.2</Emphasis> promoter is an essential and sufficient cis-acting element in response to MeJA treatment

The expression of Arabidopsis PDF1.2 gene isregulated by jasmonic acid (JA) and ethylene (ET). It also has been well documented that GCC box is an element responsive to ET, however, the responsive mechanism of JA in such plant defense gene expression is unclear. In this paper, the authors define the essential cis-acting element in PDF1.2 promoter responsive to methyl jasmonate (MeJA) through fragment deletions and site-directed mutageneses combiningAgrobacterium-mediated transient reporter gene expression in tobacco leaves. Firstly, the MeJA inducible expression o fPDF1.2 was confirmed by using the upstream -1.86 kb fragment of PDFI.2 gene. Secondly, the upstream -300— -243 bp fragment of the promoter was evidenced to respond to MeJA. To further characterize this promoter region, three point mutations were introduced into the -300— -243 bp fragment of the promoter. This result showed that the mutation of GCC box abolished MeJA induction, whereas the mutations of the G box-like and the imperfect palindrome sequence did not significantly decrease MeJA inducible effect, indicating that GCC box in PDFI.2 is essential for MeJA induction. The sufficient responsiveness to MeJA of this GCC box was further investigated by 4xGCC fused upstream to the CaMV 35S minimal promoter. This result suggested that the fused promoter was able to activate reporter gene expression in response to MeJA. Thus these results indicate that the GCC box in PDFI.2 is an essential and sufficient element to confer MeJA induction.     

rDNA介导的多拷贝整合表达载体的构建及其在酿酒酵母工业菌株中的应用

根据酵母整合质粒的设计要求,PCR扩增特定的2．2kb rDNA片段,并以此替换酿酒酵母(Saccharomyces cereristae)整合载体YIp5的URA3片段;在此基础上,引入G418抗性基因KanMX和酵母磷酸甘油激酶(phosphoglycerale kinase,PGK)组成型强启动子和终止子序列(PGKp-t),构建适合酿酒酵母工业菌株高拷贝整合表达载体pYMIKP,以细菌木糖异构酶(xylose isomerase,XI)基因xy/A为目标基因,通过载体pYMIKP引入到酵母工业菌株NAN-27中,酵母转化子在非选择培养条件下,连续生长50世代质粒稳定性为99．72％,目标基因高拷贝重组菌的木糖异构酶比酶活是对照菌株的67．2倍,达到0．672U／mg蛋白,实现了外源基因在酿酒酵母工业菌株中的稳定高效表达。     

Transgenes in F4 pMThGH-transgenic common carp (Cyprinus carpio L.) are highly polymorphic

To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F₄ generation of pMThGH transgenic common carp (Cyprinus carpio L.) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate thatMThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F₄ generation of transgenic carp are highly polymorphic. Two DNA fragments concerning integration site of transgenes were cloned from recovered transgenes, and found to be homologous to the 5′UTR ofβ-actin gene of common carp and mouse mRNA for receptor tyrosine kinase (RTK), respectively.     

Aberrant activity of Wnt/Frizzled signaling pathway in renal cancer cell lines

The expression ofWnt, Wntreceptor-Frizzled, and several other key components in Wnt pathway in renal cancer cell lines was studied. The result of semi-quantitative RT-PCR has shown that the expression level ofWnt5A andhFz5 mRNA were higher in renal cancer cell line (GRC-1) than in normal renal cell line (HK-2). This result has been confirmed byin situ hybridization. The expression of β-catenin protein was obviously higher in GRC-1 than in HK-2 (P< 0.01), but there were no different expressions of its mRNA between 3 lines. The reasons of the overexpression of β-catenin has been investigated by means of immunocytochemistry, SSCP and so on, no mutation ofβ-catenin gene and APC were found. That means that the overexpression ofWnt5A/hFz5 might be the reason of overexpression of β-catenin. It was concluded that the aberrant activity of Wnt pathway might play an important role in renal cell carcinoma.     

Cloning and sequencing of cDNA fragment of gene of wheat (Triticum aestivum. L) induced by dehydration

cDNA fragment of the gene (dehydration induced,di1) of wheat (Triticum aestivum. L) induced by 30% PEG-6000 (−1.13 MPa) treatment was isolated with mRNA differential display technique. Northern blot analysis showed that the expression ofdi1 gene improved at 10 h reached the highest at 48 h under 30% PEG-6000 treatment. cDNA fragment ofdi1 gene has been cloned and sequenced (211 bp). DNA sequence analysis shows that there is no homologue in GenBank todi1 cDNA.     

Regulatory role of the sequences downstream fromnodD3 P1 promoter ofRhizobium meliloti

The 660 bp region betweennodD3 P1 promoter and the following coding region ofRhizobium meliloti has been studied. This region is designated “downstream sequences”. It consists of two potential open reading frames, ORF1 and ORF2. Studies on the role of the downstream sequences on the activity ofnocD3 P1 with nod D3(P1)-IacZ fusion show that deletion of the sequences containing ORF2 causes the increase of the activity of the fusion; on the contrary, addition of extra copies of ORF2 markedly decreases the activity of the fusion. These results indicate that the product of ORF2 plays a negative role in the expression ofnod D3.     

Evolutionary mode of metallothioneins inferred from Cd-MT genes of Tetrahymena

FromTetrahymena thermophila (strain BF5), the coding region ofCd-MT gene was cloned and sequenced, and identified as MTTI isoform. A serial duplicate structure is discovered in its amino acid sequence, which separates the coding region into three parts (Part 1: 7–61: Part 2: 64–118; Part 3: 122–162). The alignments among them and comparison with the corresponding parts of MT1 isoform suggest that MT1 and MTT1 isoforms both come from the same ancient gene that is homologous to Part 1, and Cd-MTs ofTetrahymena are aroused by such ancient gene duplication. The prediction of secondary structures and the analysis of the disulfide-bonding state of cysteine show that there are a lot of differences between MT1 and MTT1 isoforms, which maybe relate to their function mechanism. Foundation item: Supported by KSCX of Chinese Academy of Sciences Grant (SW-102) and the Allocation from the Earmaarked Grant for Hong Kong. Biography: WAN Mingliang (1980-). male, Ph. D. candidate, research direction: molecular ecotoxicology of protozoan.     

Characterization of the flagellar biosynthesis regulatory geneflbD inAzospirillum brasilense

A flagellar gene cluster fragment includingflbD ofAzospirillum brasilense was cloned and sequenced. TheflbD mutant strain was found to be nonmotile—losing both polar and lateral flagella (Fla⁻Laf⁻). Motility and flagella were regained by complementation with plasmid-borne multicopyflbD, but altered with larger swarming circle and fewer lateral flagella on the semisolid plate. This result indicated that FlbD plays an important role in the regulation of both polar and lateral flagellar biosynthesis inA. brasilense.     

Cloning, construction of prokaryotic expression vector and expression ofEscherichia coli cytosine deaminase gene

Cytosine deaminase gene ofEscherichia coli strain H-30 was cloned, and its initiation codon of ‘GTG’ was mutated to ‘ATG’ by PCR. Prokaryotic recombinant expression vector pBV220-CD was constructed. Clone with high enzyme activity were selected by detecting their specific activity of cytosine deaminase. 5-FC(5-FC, 5-fluorocytosine) could induce the lethal toxicity to cells containing active CD gene. DNA sequence analysis indicated that there were 16 altered bases and 5 of them resulted in the alteration of amino acids in predicted peptide by comparing DNA sequence of the clone H-30-CD-11 with high enzyme activity with CD gene reported in Gene Bank.     

A Note on the Prime Spectrums of Character Rings of Finite Groups

0Introduction LetGbeafinitegroupoforder|G|=g,andletR(G)denotethecharacterringofG,whichisgeneratedbyalltheirreduciblecomplexcharactersofG.LetZbetherationalintegerringandNthesetofnatural numbers,andletZ[ω]betheintegralextensiongeneratedbyaprimitiveg throotωofunity.SupposethatSisasubringof thealgebraicnumberfieldsuchthatZ[ω]S.πisasetofra tionalprimenumbersdefinedasfollowsπ={p｜pisarationalprimenumbersuchthatp-1S}.Definition1WecallthataconjugacyclassCofthefinite groupGisaπregularconju…     

Gene mapping of a new coat mutation in mouse

Gene mapping of a mouse coat mutation has been investigated. First, 100 10-bp random primers were used to amplify DNA, but the mutation could not be located by this method because there were no correlation between the amplified products and coat phenotypes. Second, by usingIdh1, Car2, Mup1, Pgb1, Hbb, Es10, Es1, Mod1, Gdc1, Ce2, Es3 as genetic markers, linkage test crosses (two-point test) consisting of intercrossing uncovered BALB/c mice (homozygotes) to CBA/N and C57BV6 mice with normal hair and backcrossing the heterozygotes of the F1 to the uncovered BALB/c mice were made. It was soon evident that the mutation was linked toEs3 on chromosome 11. Furthermore, three-point test was made by usingEs3 and D11Mit8 (a microsatellite DNA) as genetic markers. The result showed that the mutation was linked toEs3 with the percentage recombination of (7.89 ± 2.19)%, and linked to Dl1Mit8 with the percentage recombination of (26.30± 3.57)%. The percentage recombination betweenEs3 and D11Mit8 was (32.90±3.81)%. The mutation was named Uncovered, with the symbolUncv. According to the recombinations, the loci order was D11Mit8-26.30±3.57-Uncv- 7.89 -2.19-Es3. From the location on the chromosome, it was concluded that the mutation was a new mutation which affected the skin and hair structure of mouse. TheUncv has entered MGD (Mouse Genome Database).     

Cloning, sequencing and function ofsanB, a gene related to nikkomycin biosynthesis ofStreptomyces ansochromogenes

A 6.0 kb DNA fragment related to nikkomycin biosynthesis was cloned from nikkomycin-producingStreptomyces ansochromogenes 7100. Sequence analysis showed that the 1.9 kbTth111 I fragment, a part of the 6.0 kb DNA fragment, contains one complete ORF designatedsanB (GenBank accession No. AF224501), which is composed of 1740 bp encoding a protein consisting of 580 amino acid residues. Its start codon is GTG at 100 bp position and stop codon is TGA at 1840-bp position. Database searching indicated that the deduced protein ofsanB is homologous to the histidinol-phosphate aminotransferase inStreptomyces coelicolor with 31% identities and 47% positives. Gene disruption was performed to study the function ofsanB. It was found that disruptants ofsanB lost the ability to synthesize nikkomycin, which reveals thatsanB is a novel gene essential for nikkomycin biosynthesis.