OBJECTIVEScreening for special genes of matrix proteins of dentin and enamel of mouse dental germ.

METHODSA cDNA library of dental germ of mouse was screened by differential display. The interesting clones were sequenced.

RESULTSSix positive clones were isolated from the cDNA library. The sequence of one of the six positive clones was homologous with the ameloblastin sequence of rat. There are 497 homologous base pairs between the 526 base pairs sequenced by pTriplEX 3' primer of this clone and the 32-580 sequence of the rat ameloblastin gene; and there are 533 homologous base pairs between the 567 base pairs sequenced by pTriplEX 5' primer of this clone and the 1285-1854 sequence of the rat ameloblastin gene.

CONCLUSIONSThe full length cDNA sequence of the mouse ameloblastin was cloned.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Gene Library ; Mice ; Molecular Sequence Data

This paper is to show a way of acqusition of the variable region gene of rabbit osteoprotegerin (OPG) and to analyse series. Total RNA was extracted from rabbit tibia, transcripted reversely into cDNA with random primers. The variable region of the OPG gene ampliflied using 5'RACE. Sequencing was confirmed by agarose gel electrophoresis and sequencing analysis. Full length of OPG gene was 1540bp that encoding 400 amino acids. It shared 89% identity with human OPG in whole amino acid sequence and about 85% with rattus norvegicus and other mammal. The OPG sequence of rabbit was obtained by 5'RACE, which could provide a good basis for OPG functional study.
Animals ; Base Sequence ; Molecular Sequence Data ; Osteoprotegerin ; genetics ; Rabbits ; Sequence Homology, Amino Acid

OBJECTIVETo identify and confirm a novel HLA allele.

METHODSA new human leukocyte antigen class I allele was found during routine HLA genotyping by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) and sequencing-based typing (SBT).

RESULTSThe novel HLA-B*52 allele was identical to B*52:01:01 with an exception of one base substitution at position 583 of exon 3 where a C was changed to T resulting in codon 195 changed from CAC(H) to TAC(Y).

CONCLUSIONA new HLA class I allele, B*52:11, is identified, and is named officially by the WHO Nomenclature Committee.

Alleles ; Amino Acid Sequence ; Base Sequence ; Genotype ; HLA-B Antigens ; genetics ; Humans ; Molecular Sequence Data ; Sequence Alignment ; Sequence Analysis, DNA

Chloramphenicol-resistant gene was cloned and analyzed by constructing genomic DNA library of Serratia marcescens KMR-3. It showed that cloned chloramphenicol-resistant gene encoded a protein product of 397 amino acids. The protein belonged to PRK10473 protein, and it showed 92% similarity to drug resistance transporter, Bcr/CflA subfamily of Serratia proteamaculans 568. Regulation elements including promoter, terminator, Shine-Dalgarno (SD) sequence and transcription start site also were identified.
Amino Acid Sequence ; Base Sequence ; Chloramphenicol Resistance ; genetics ; Cloning, Molecular ; Molecular Sequence Data ; Serratia marcescens ; classification ; genetics

4-coumarate coenzyme A ligase is a key enzyme of phenylpropanoid metabolic pathway in higher plant and may regulate the biosynthesis of ferulic acid in Angelica sinensis. In this study, the homology-based cloning and rapid amplification of cDNA ends (RACE) technique were used to clone a full length cDNA encoding 4-coumarate coenzyme A ligase gene (4CL), and then qRT-PCR was taken for analyzing 4CL gene expression levels in the root, stem and root tissue at different growth stages of seedlings of A. sinensis. The results showed that a full-length 4CL cDNA (1,815 bp) was obtained (GenBank accession number: KT880508) which shares an open reading frame (ORF) of 1 632 bp, encodes 544 amino acid polypeptides. We found 4CL gene was expressed in all tissues including leaf, stem and root of seedlings of A. sinensis. The expressions in the leave and stem were increased significantly with the growth of seedlings of A. sinensis (P < 0.05), while it in the root showed little change. It indicates a time-space pattern of 4CL gene expression in seedlings of A. sinensis. These findings will be useful for establishing an experiment basis for studying the structure and function of 4CL gene and elucidating mechanism of ferulic acid biosynthesis and space-time regulation in A. sinensis.
Amino Acid Sequence ; Angelica sinensis ; genetics ; Base Sequence ; Cloning, Molecular ; Coenzyme A Ligases ; genetics ; DNA, Complementary ; chemistry ; Molecular Sequence Data

It was constructed that a genomic DNA library from Lactobacillus sp. MD-1 yielding D, L-lactic acid. The gene encoding L-lactate dehydrogenase (L-LDH) was cloned from the genomic library of strain MD-1 by complementation in E. coli FMJ144 which was lactate dehydrogenase and pyruvate-formate lyase double defective mutant. The nucleotide sequence of the ldhL gene predicted a protein of 316 amino acid starting with ATG. The putative molecular weight of the L-LDH amino acid sequence was 33.84kD. A putative typical promoter (-35 and -10 boxes) had been observed in the 5' noncoding region. An rho-independent transcriptional terminator has been observed in the 3' noncoding region. Three highly conserved regions (Gly13 approximately Asp50, Asp73 approximately Ileul00 and Asn123 approximately Arg154) with several conserved residues had been identified. Gly13 approximately Asp50 was NADH-binding site domain. Asp73 approximately Ileu100 and Asn123 approximately Arg154 were reported to be the active site domains. The ldhL and the L-LDH of Lactobacillus sp. MD-1 showed the low identity and similarity with other Lactobacilli, and the highest percentage were 61.9% and 68.9% respectively. All the above indicated this gene is a novel ldhL.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; L-Lactate Dehydrogenase ; chemistry ; genetics ; physiology ; Lactobacillus ; genetics ; Molecular Sequence Data

The nucleotide sequence and N-, C-terminal amino acid sequences of alpha,beta-subunit of glutaryl 7-ACA acylase C130 from Pseudomonas sp. 130 were determined. The alignment of the acylase C130 with the other acylases shows that it has high homology with the acylases from Pseudomonas sp. GK16 and C427, but low homology with the others. There is large difference in the N-terminal of alpha-subunit, while the N-terminal of beta-subunit has significant conservation.
Amino Acid Sequence ; Base Sequence ; DNA, Bacterial ; analysis ; Genes, Bacterial ; Molecular Sequence Data ; Penicillin Amidase ; genetics ; Pseudomonas ; enzymology ; genetics ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid

OBJECTIVETo study the EV71 Chinese strain SHZH98 and analyze its genetic evolution using 3c gene as index.

METHODSThe 3C gene cDNA of EV71 Chinese strain SHZH98 was amplified by PCR, the PCR product was sequenced.

RESULTSThe EV71 Chinese mainland strain SHZH98 3C segment was 549 bps in length. Comparison of nucleotide sequences from other enteroviruses which have been published, revealed a higher homology to strain MS, 78.7% at nucleotide level and 93.45% at deduced amino acid level. The homology to strain BrCr was 76.7% at nucleotide level and 89.1% at deduced amino acid level. Taiwan strains POLY,NCKU,TW2086,TW2272 shared a lower homology with Chinese mainland strain SHZH98, 74.0%, 73.8%, 71.9%, 69.8% at nucleotide level and 90.7%, 90.2%, 84.2%, 82.5% at deduced amino acid level. The genetic progress analysis revealed that EV71 Chinese mainland strain SHZH98 3C segment shares more homology with European and American strains than Taiwan strains.

CONCLUSIONThe non-structural protein of EV71 Chinese strains may have different evolutionary process from Taiwan strains.

Amino Acid Sequence ; Base Sequence ; Enterovirus ; genetics ; isolation & purification ; Genes, Viral ; Humans ; Molecular Sequence Data ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Homology, Amino Acid ; Sequence Homology, Nucleic Acid

Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferase II (NPT II) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work, by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc + was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.
Amino Acid Sequence ; Base Sequence ; Drug Resistance ; genetics ; Genetic Markers ; Genetic Vectors ; Kanamycin Kinase ; genetics ; Molecular Sequence Data ; Mutation

To better understand the genomic structure and function of Bombyx mori bidensovirus (China isolate) VD1, the VD1 was purified and cloned into the pUC119 vector, and the complete nucleotide sequence of VD1 was determined. Sequence analysis showed that VD1 genome consisted of 6543 nts including inverted terminal repeats (ITRs) of 224 nts. In the viral genome, three major open reading frames (ORF1, ORF2 and ORF3) in the plus strand and one major ORF (ORF4) in the complementary strand were identified. Comparison of the complete genome sequence between Bombyx mori bidensovirus (China isolate) and BmDNV-2 (Yamanashi isolate) showed an identity of 98.4% in VD1, with a total number of 104 bp substitutions and 1 bp insertions found in Bombyx mori bidensovirus (China isolate), the highly variable regions were mainly located in VD1 ORF3 and VD1 ORF4. Northern blotting revealed that VD1 contained 1.1 kb and 1.5 kb transcript in the left-half 'plus' strand, and one transcript about 3.3 kb of 'minus' strand in the right-half. Sequencing of 3' and 5' ends of transcript products showed the 1.1 kb transcript started at nt 290 and ended at nt 1437, the 1.5 kb transcript was found to start nt 1423 and ended at 2931, and the 3.3 kb transcript was found to start nt 6287 and ended at nt 2922. Therefore, the 1.5 kb transcript in the left-half plus' strand and 3.3 kb transcripts of minus' strand in the right-half overlapped for 10 nts at the 3' ends. These results indicate that this virus employs a transcription strategy that is radically different from that of the other reported DNVs.
Amino Acid Sequence ; Animals ; Base Sequence ; Bombyx ; virology ; Densovirus ; genetics ; Genome, Viral ; Molecular Sequence Data ; Transcription, Genetic