Molecular Cloning and Sequence Analysis of IGF-Ⅰfrom Triangular Bream (Megalobrama terminalis)

The insulin-like growth factor Ⅰ(IGF-Ⅰ) gene of triangular bream (Megalobrama terminalis)(GenBank No.AY247412)(Tb)was cloned for the first time from liver by RT-PCR. Thenucleotide sequence analysis showed the Tb IGF-ⅠcDNA consisted of 486 nucleotides andencoded 117 amino acids including B, C, A, D and E five domains. Analysis of E-domainindicated that cloned Tb IGF-Ⅰbelonged to IGF-ⅠEa-2 subtype. Identity analysis showedthe IGF-Ⅰnucleotide sequence shared 99.8% homology with bluntnose bream, 88.8% withgrass carp, 85.8% with common carp; the pre-IGF-Ⅰamine acid sequence shared 99.4% withbluntnose bream, 88.8% with grass carp, 85.4% homology with common carp. In the CyprinusCarpio, the higher homology of nucleotide sequence and amino acid sequence in IGF-Ⅰshowed that the closer relationship the fishes have. These results could provide basicdata for the research on Tb germplasm and the development and utilization of biologicalfeed additives.

Molecular cloning, characterization and expression analysis of a catalase gene in Paphia textile

Catalase is an important antioxidant protein that can protect organisms against various forms of oxidative damage by eliminating hydrogen peroxide. In this study, the catalase c DNA of Paphia textile（Pt CAT） was cloned using RTPCR and rapid amplification of c DNA ends（RACE）. Pt CAT is 1 921 bp long and consists of a 5′-UTR of 50 bp, a 3′-UTR of 349 bp, and an ORF of 1 542 bp that encodes 513 amino acids with a molecular weight of 58.4 k D and an estimated isoelectric point of 8.2. Sequence alignment indicated that Pt CAT contained a highly conserved catalytic signature motif（^（61）FNRERIPERVVHAKGAG^（77））, a proximal heme-ligand signature sequence（^（352）RLFSYSDP^（359））, and three catalytic amino acid residues（H^（72）, N^（145）, and Y^（356））. Pt CAT also contains two putative N-glycosylation sites（^（34）NKT^（36） and ^（437）NFT^（439）） and a peroxisome-targeting signal（^（511）AQL^（513））. Furthermore, Pt CAT shares 53%–88% identity and 29%–89% similarity with other catalase amino acid sequences. Pt CAT m RNA was present in all tested organs, including the heart, digestive gland, adductor muscle, gonad, gill, and mantle, but its expression was highest in the digestive gland. High-temperature-induced stress produced two expression patterns of Pt CAT m RNA： first, an initial up-regulation followed by a down-regulation in the heart, digestive gland, and gonad and, second, consistent down-regulation in all other organs. These results demonstrate that Pt CAT is a typical member of the catalase family and might be involved in the responses to harmful environmental factors.

Molecular Cloning of Myostatin Partial cDNA of Beijing Duck and Its Expression in Breast Muscle

In this experiment, 500 bp cDNA of myostatin gene was cloned from a Beijing duck＇s breast, The duck myostatin gene was found to have 98, 96, 95, 88, and 87% sequence similarity at the cDNA level with domestic goose, chicken, domestic pigeon, human, and pig, respectively. The predicted amino acid sequence has an overall similarity with a comparable region of turkey 99%, domestic goose 98%, and chicken 99%. Conserved domains of deduced amino acids showed that it belonged to the TGF-beta family. Myostatin expression in breast muscle was higher at 28, 35, and 42 days than at 7, 14, and 21 days. The pattern of myostatin expression was closely parallel to the trend of breast muscle growth, suggesting that myostatin might play an important role in breast muscle development. It was possible to postulate that myostatin may be a major determinant of muscle mass in breast muscle, as shown in other species.

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction (PCR) technique. We found that 2 and 3 mAbs utilized genes of the VHIV and VHIII families, respectively. The former 2 VH segments were in germline configuration. A common VH segment, with the best similarity of 90.1 % to the published VHIII germline genes, was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs. This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHIII gene. All these polyreactive mAbs displayed a large NDN region (VH-D-JH junction). The entire H chain V regions of these polyreactive mAbs are unusually basic. The analysis of the charge properties of these mAbs as well as those of other poly- and mono- reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites (CDRs), may be an important structural feature involved in antibody polyreactivity.

Rapid Amplification of Flanking Sequences of a Known DNA Region by Partial Restriction Digestion and Hot Start PCR

A simple and efficient method for cloning the flanking genomic sequences of a known DNA region is reported in this study. This method combined partial restriction endonuclease digestion, adaptor ligation, and a single round polymerase chain reaction. Total genomic DNA was partially digested with the frequent-cutting restriction enzyme Mse I. The partially digested products were ligated to an unphosphorylated adaptor. A hot start PCR amplification with Taq polymerase and dNTP was performed with a DNA-specific primer and the adaptor primer complementary to the adaptor and the Mse I recognition site. The amplified products were fractionated, cloned and sequenced. By this method, we cloned the downstream region of a gynoecious marker TG/CAC234 from cucumber （Cucumis sativus L.）.

Isolation and sequence analysis of a Dof protein gene(PtDof1) in Populus

Both cDNA and DNA clones of PtDof1 （GenBank Accession No. FJ402844 and FJ402845） were isolated from plants grown in tissue culture ofPopulus tornentosa. The DNA sequence is 1597 bp including two exons and one intron. The cDNA is 969 bp in length with a 765 bp open reading frame which is capable of encoding 255 amino acids. The deduced amino acids sequence of the PtDofl protein shares 65%, 56% and 55% identity with Vitis vinifera （CAO48618）, Nicotiana tabacum （CAA08755） and Glycine max （ABI 16022） Dof protein by blast analysis in GenBank. Phylogenic analysis suggests PtDof1 gene could belong to the Dofgene family. PtDofl protein contains an unusual conserved single zinc finger with the pattern of C-X2-C-X21-C-X2-C, which may play a functional role in tissue-specific expression and possibly the auxin response of endogenous plant genes.

THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

Complete genome sequence analysis of two Citrus tatter leaf virus(CTLV) isolates from China

In order to understand molecular characterization of Citrus tatter leaf virus（CTLV） isolated from China,full-length cDNAs of CTLV-MTH and CTLV-XHC from Citrus reticulata and Citrus sinensis were cloned and sequenced based on whole-genome amplification by RT-PCR.The complete nucleotide sequences of CTLV-MTH and CTLV-XHC were determined to be 6497 nucleotides in length and shared 79.9-91.0%and 78.8-98.0%nucleotide sequence identity,respectively,with other Apple stem grooving virus（ASGV） or CTLV strains available in GenBank.Unexpectedly,CTLV-MTH showed the highest nucleotide sequence identity（91%） with an apple isolate of ASGV,followed by 86.5%with ASGV-HH and 85.7%with ASGV-CHN.Furthermore,CTLV-MTH and three ASGV strains were grouped to a separate cluster in the phylogenetic tree,suggesting it has a closer relationship to ASGV than to CTLV.Therefore,it can be concluded roughly that CTLV may be not a distinct strains of ASGV.We proposed that Citrus tatter leaf virus should be renamed Apple stem grooving virus.

Effect of methamidophos on soil fungi community in microcosms by plate count,DGGE and clone library analysis

Methamidophos was widely used a pesticide in northern China. The potential influences of methamidophos on soil fungal community in black soil were assessed by plate count, 28S rDNA-PCR-DGGE, and clone library analysis. Three methamidophos levels （50, 150, and 250 mg/kg） were tested in soil microcosms. Results from plate count during a 60-d microcosm experiment showed that high concentrations of methamidophos （250 mg/kg） could significantly stimulate fungal populations. DGGE （denaturing gradient gel electrophoresis） fingerprinting patterns showed a significant difference between the responses of culturable and total fungi communities under the stress of methamidophos. Shannon diversity indices calculated from DGGE profiles indicated that culturable fungi in all microcosms with methamidophos treatment increased after 1 week of incubation. However, the diversity indices of total fungi decreased in the first week, as compared to the stimulation of culturable fungi. At the 8th week, however, all the microcosms treated by methamidophos were similar to the control microcosms in community structure as suggested by the Shannon diversity indices for both culturable and total fungi. In contrast, after 1 week the fungal structure of culturable and unculturable both were disturbed to different extent under the stresses of methamidophos by clustering analysis. Clone sequencing analysis indicated the stimulation of pathogenic and unculturable fungal populations by methamidophos treatment, suggetsing potential risks of plant disease outbreak.

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites

To our knowledge,few reports on Demodex studied at the molecular level are available at present.In this study our group,for the first time,cloned,sequenced and analyzed the chitin synthase(CHS) gene fragments of Demodex folliculorum,Demodex brevis,and Demodex canis(three isolates from each species) from Xi'an China,by designing specific primers based on the only partial sequence of the CHS gene of D.canis from Japan,retrieved from GenBank.Results show that amplification was successful only in three D.canis isolates and one D.brevis isolate out of the nine Demodex isolates.The obtained fragments were sequenced to be 339 bp for D.canis and 338 bp for D.brevis.The CHS gene sequence similarities between the three Xi'an D.canis isolates and one Japanese D.canis isolate ranged from 99.7% to 100.0%,and those between four D.canis isolates and one D.brevis isolate were 99.1%-99.4%.Phylogenetic trees based on maximum parsimony(MP) and maximum likelihood(ML) methods shared the same clusters,according with the traditional classification.Two open reading frames(ORFs) were identified in each CHS gene sequenced,and their corresponding amino acid sequences were located at the catalytic domain.The relatively conserved sequences could be deduced to be a CHS class A gene,which is associated with chitin synthesis in the integument of Demodex mites.

c D N Acloning and sequence analysis of hepatitis G virus genome isolatedfrom a Chinese blood donor

Objective To obtain full length sequence of a Chinese hepatitis G virus (HGV) strain (HGVch) and investigate the genetic characteristic of HGVch and its identity to other isolates Methods Reverse transcription (RT) and nested PCR were used to screen HGV RNA positive serum and amplify cDNA fragments A positive serum without known hepatitis virus markers was selected for isolating HGV RNA template The HGV genome was divided into 12 overlapping fragments and directly cloned into pGEM T vector Sequences were determined by dideoxy terminus end method of DNA sequencing and then analyzed by computer Results The twelve fragments of HGVch cover 9213 nucleotides in length, containing a large open reading frame (ORF) encoding 2873 animo acids polyprotein that began with a methonine residue and ended at termination codon HGVch is about 86 5%-89 5% identical to other known HGV isolates at the nucleotide level and about 93 9%-96 2% at the deduced animo acid level Conclusion HGV is a non A E hepatitis causal agent, proved to be related with posttransfusion hepatitis in all over the world Chinese HGV isolate has very close relationship to other isolates from Africa, Europe, Japan, without significant difference across the entire genome It is suggested that the sequences of HGV isolates are very conservative and the evolution is very slow

Genetical Study of Mutation in Maternal-fetal ABO Incompatibility

This study looked into a family involving a rare mother-child ABO blood type inconsistency and explored its genetic and molecular basis. In the family, the mother had type AB blood and the father was blood type B and they gave birth to a baby of blood type O. Their blood types were phenotypically identified by using different techniques, including micro-column gel test, immune inhibition test, absorption and elution tests. The sequences of all 7 exons of ABO allele from the core family members were determined by using PCR and clone-based sequencing. The loci of mutated gene were compared against normal human genes. The result showed that the mother＇s erythrocytes were agglutinable with monoclonal anti-A antibody（2＋） and had agglutination reaction with anti-B antibody（4＋）. The mother＇s serum registered agglutination action with standard blood type A cells. The findings showed an ABO inconsistency. When domestic antibodies were used, the mother＇s erythrocytes yielded agglutination reaction with humanized anti-B serum（4＋） and anti-B monoclonal antibody but were non-agglutinable with humanized anti-A serum and anti-A monoclonal antibody. Upon absorption and elution, the titer of anit-A antibody was 128 both before and after the absorption test, with no significant difference found between pre- and post-absorption values. Our results confirmed that the mother＇s allelic gene was type B and contained type A. The father＇s blood type was type B, and son＇s blood type was type O. Clone-based sequencing revealed that the mother carried a heterozygous gene of B101.01（nt A640→G）/O01, which contained an M214→V mutation that could express a weak expression of antigen A, resulting in blood type AB. However, their son did not have the M214→V mutation, which yielded a false ABO-inconsistency between him and his mother. We were led to conclude that type B gene with a M214→V mutation can encode both antigen B and weak antigen B that can lead to false ABO-inconsistencies.

Difference in DNA sequences in SSU rDNA variable regions among pathogens isolated from different epidemic foci of visceral leishmaniasis in China

To confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L d ) isolates from different epidemic foci in China Methods Specific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM R T Easy Vectors After that, the specific fragments were sequenced by an automated DNA sequencer Results Sequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length All 5 point mutations were located in two unique sequence blocks (UQ Ⅰ and UQ Ⅱ), and no insertions or deletions were found The identities of comparison of Leishmania in GeneBank were more than 98% Conclusion Five point mutations exist in the SSU rDNA variable region of 5 L d isolates from different epidemic foci of visceral leishmaniasis (VL) in China Sequence differences of the SSU rDNA variable region exist among L d isolates from different foci

Sequence-length variation of mtDNA HVS-I C-stretch in Chinese ethnic groups

The purpose of this study was to investigate mitochondrial DNA (mtDNA) hypervariable segment-I (HVS-I) C-stretch variations and explore the significance of these variations in forensic and population genetics studies. The C-stretch sequence variation was studied in 919 unrelated individuals from 8 Chinese ethnic groups using both direct and clone sequencing ap-proaches. Thirty eight C-stretch haplotypes were identified, and some novel and population specific haplotypes were also detected. The C-stretch genetic diversity (GD) values were relatively high, and probability (P) values were low. Additionally, C-stretch length heteroplasmy was observed in approximately 9% of individuals studied. There was a significant correlation (r=-0.961, P<0.01) between the expansion of the cytosine sequence length in the C-stretch of HVS-I and a reduction in the number of up-stream adenines. These results indicate that the C-stretch could be a useful genetic maker in forensic identification of Chinese populations. The results from the Fst and dA genetic distance matrix, neighbor-joining tree, and principal component map also suggest that C-stretch could be used as a reliable genetic marker in population genetics.

Cloning and sequencing of 23 kDa membrane protein of Chinese Schistosoma japonicum

Abstract Objective To develop the vaccine of Chinese Schistosomiasis japonicum, we try to prepare the 23kDa membrane protein of Schistosoma japonicum Chinese strain with the gene cloning techniques. Methods A pair of primers P1 and P2 was systhesized according to the DNA sequence of the 23kDa membrane protein of Schistosoma mansoni, a BamH1 site was added at 5' end of the primer P1 and the Sall site was added at the 5' end of the primer P2. The gene DNA fragment of the 23kDa membrane protein (SjC23) of Schistosoma japonicum was amplified from the cDNA library of Schistosoma japonica by PCR, the purified target DNA fragment was inserted into the vector pUC18/19 to form the recombinant, and sequenced in Livopool University, UK and Fudan Universtiy, China respectively. The DNA sequence was analyzed with Dnasis software, and the amino sequence was deduced with the SWISS PORT software. Results The size of DNA of 23kDa membrane protein of Schistosoma japonica Chinese strain (SjC23) was 657bp, and it was the same size as that of Sm23 and Sj23 Philippine strain. The DNA sequence of Sj23 Chinese strain (SjC23) was 100% in homology with the SjC23 Philippine strain, and 79.5% in homology with Sm23. The deduced amino acid sequence of SjC23 was 84% in homology with the Sm23, and 100% in homology with Philippine strain. There were two hydrophilic domains in the SjC23, one was located at the N terminal (amino acid 36-56), and another was at the C terminal (amino acid 108-183). Conclusions The gene of the 23kDa membrane protein of Schistosoma japonica Chinese strain has been cloned, and this work has laid the foundation for the development of the vaccine of Schistosoma japonica Chinese strain.

Continuous desulfurization and bacterial community structure of an integrated bioreactor developed to treat SO_2 from a gas stream

Sulfide dioxide（SO2） is often released during the combustion processes of fossil fuels. An integrated bioreactor with two sections, namely, a suspended zone（SZ） and immobilized zone（IZ）, was applied to treat SO2 for 6 months. Sampling ports were set in both sections to investigate the performance and microbial characteristics of the integrated bioreactor. SO2 was effectively removed by the synergistic effect of the SZ and IZ, and more than 85%removal efficiency was achieved at steady state. The average elimination capacity of SO2 in the bioreactor was 2.80 g/（m3·hr） for the SZ and 1.50 g/（m3· hr） for the IZ. Most SO2 was eliminated in the SZ. The liquid level of the SZ and the water content ratio of the packing material in the IZ affected SO2 removal efficiency. The SZ served a key function not only in SO2 elimination, but also in moisture maintenance for the IZ. The desired water content in IZ could be feasibly maintained without any additional pre-humidification facilities. Clone libraries of 16 S r DNA directly amplified from the DNA of each sample were constructed and sequenced to analyze the community composition and diversity in the individual zones.The desulfurization bacteria dominated both zones. Paenibacillus sp. was present in both zones, whereas Ralstonia sp. existed only in the SZ. The transfer of SO2 to the SZ involved dissolution in the nutrient solution and biodegradation by the sulfur-oxidizing bacteria.This work presents a potential biological treatment method for waste gases containing hydrophilic compounds.

Construction of an infectious cDNA clone of Tembusu virus isolated from breeder Peking ducks

Dear Editor,Since April 2010,an outbreak of a new disease has elicited symptoms of high fever,loss of appetite,and reduction in egg production in layer ducks in eastern China;this phenomenon has now spread throughout China(Cao et al.,2011;Su et al.,2011).The causative agent of the disease was identified as Tembusu virus(TMUV),which was classified into the genus Flavivirus,