DNA sequence : the journal of DNA sequencing and mapping最新文献

Adenine phosphoribosyltransferase (APRT) is the key enzyme that converts adenine to adenosine monophosphate (AMP) in the purine salvage pathway. It was found that several different forms of APRT gene exist in plants, but no APRT gene in maize has been reported up to now. In this study, a novel maize APRT gene was cloned and characterized through a combination of bioinformatic, RT-PCR and RACE strategies. The full length of APRT cDNA sequence is 1202 nucleotides, with an ORF encoding 214 amino acid residues. Alignment of the deduced protein with that of other plant APRT genes indicates that the new gene is the form 2 of maize APRT, thus it was named ZmAPT2. Through basic local alignment search tool, search in the genomic survey sequence database of MaizeGDB, the putative genomic sequence of ZmAPT2 was obtained. Comparison of the cDNA and genomic sequence of the ZmAPT2 gene revealed that it contained seven exons and six introns. The locations of the introns within the maize ZmAPT2 coding region were consistent with those in the previously isolated APRTs of arabidopsis and rice. RT-PCR analysis showed that ZmAPRT was constitutively expressing in different organs under high temperature and salt stresses. Southern blot analysis indicated that at least three APRT genes existed in maize genome. These results confirmed that the novel maize ZmAPT2 gene was truly identified, and its potential role in maize growth and development was discussed.

The complete mitochondrial DNA sequence of the frigate tuna Auxis thazard and two divergent mitotypes (Mitotype I and Mitotype II) of the bullet tuna Auxis rochei have been determined. The total length of the mitogenomes was 16,506, 16,501 and 16,503 bp, respectively. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs and 22 tRNAs) and organization similar to those observed in most other vertebrates. The major non-coding region (control region) ranged between 843 and 847 bp in length, and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Auxis with respect to other tuna fish. Molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.

Simple sequence repeats (SSRs) are omnipresent in prokaryotes and eukaryotes, and are found anywhere in the genome in both protein encoding and noncoding regions. In present study the whole genome sequences of seven chromosomes (Shigella flexneri 2a str301 and 2457T, Shigella sonnei, Escherichia coli k12, Mycobacterium tuberculosis, Mycobacterium leprae and Staphylococcus saprophyticus) have downloaded from the GenBank database for identifying abundance, distribution and composition of SSRs and also to determine difference between the tandem repeats in real genome and randomness genome (using sequence shuffling tool) of the organisms included in this study. The data obtained in the present study show that: (i) tandem repeats are widely distributed throughout the genomes; (ii) SSRs are differentially distributed among coding and noncoding regions in investigated Shigella genomes; (iii) total frequency of SSRs in noncoding regions are higher than coding regions; (iv) in all investigated chromosomes ratio of Trinucleotide SSRs in real genomes are much higher than randomness genomes and Di nucleotide SSRs are lower; (v) Ratio of total and mononucleotide SSRs in real genome is higher than randomness genomes in E. coli K12, S. flexneri str 301 and S. saprophyticus, while it is lower in S. flexneri str 2457T, S.sonnei and M. tuberculosis and it is approximately same in M. leprae; (vi) frequency of codon repetitions are vary considerably depending on the type of encoded amino acids.

The present study was carried out to characterize the DGAT1 gene of Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and DGAT1cDNA were synthesized by RT-PCR, then cloned using pDRIVE cloning vector and sequenced. The sequencing revealed that the size of DGAT1 gene was 1470 bp with GC content of 62.30%. The gene encoded for 489 amino acid precursors and that it possessed 32 amino acids signal peptide. The similarity of buffalo DGAT1 mRNA sequence with that of cattle, pig, monkey, human, mice and rat were determined as 98.4, 90.7, 85.4, 85.0, 77.4 and 77.1%, respectively. Phylogenetic tree constructed from the derived DGAT1 protein sequences of 15 different species illustrated a unique branches for mammals, fly, nematode and plants. Among mammals, cattle and buffalo grouped together, whereas swine formed another group in the same branch. Four motifs were predicted in buffalo DGAT1 peptide sequence, one N-linked glycosylation site (246th position), two putative tyrosine phosphorylation site (316 and 261), one putative diacylglycerol binding site (382-392 amino acid position) and a conserved domain MBOAT (membrane bound acyl transferase from 150 to 474 amino acids) with a histidine as an active residue.

Glucose-6-phosphatase is a multicomponent enzyme composed of a transporter subunit and a catalytic subunit that is involved in hepatic glucose production. The objective of the present study was to determine the complete nucleotide sequence of feline hepatic glucose-6-phosphatase catalytic subunit (G6Pc) cDNA and to perform comparative analysis of the molecular features of the feline G6Pc cDNA and protein. Feline G6Pc cDNA contains 2261 bases and encodes a 357 aa protein. The feline cDNA and protein are highly conserved with overall identity ranging from 73-86% to 86-95%, respectively, among mammalian species. Membrane topology, phosphatase consensus sequence, ER retention sequence, N-glycosylation sites and active site residues are conserved in the feline protein. Analysis of the putative feline G6Pc protein did not reveal any species-specific features to explain the unusual in vivo regulation of G6Pase activity reported in feline liver.

Two cellulase cDNAs (GHF10-Pc1 and GHF10-Pc3) belonging to glycoside hydrolase family 10 (GHF10) were successfully isolated and characterized from stomach tissue of golden apple snail (Pomacea canaliculata), a kind of herbivorous mollusca. Sequencing analysis revealed full-length cDNAs of 1300 and 1277 bp in length, respectively. The open reading frame (ORF) of cellulase cDNA was 1188 and 1191 bp, encoding 395 and 396 amino acid, respectively. Sequence alignment revealed that GHF10-Pc1 and GHF10-Pc3 shared high identity with glycosyl hydrolase family 10 (GHF10) and had an overall similarity of 98 and 82% to those of Ampullaria crossean cellulase EGX. A neighbour-joining tree showed a clear differentiation between each species and also indicated that GHF10-Pc1 and GHF10-Pc3 from P. canaliculata and A. crossean EGX are closely related phylogenetically. The genomic organization of cellulase GHF10-Pc1 and GHF10-Pc3 genes was also investigated. The GHF10-Pc1 and GHF10-Pc3 genes spanned over 4937 and 4512 bp, respectively. Both genes contained 9 exons interrupted by eight introns. The result verified the endogenous origin of the GHF10-Pc1 and GHF10-Pc3 genes. Analysis of RNA by RT-PCR from several ages of P. canaliculata revealed that neither gene was expressed in eggs. GHF10-Pc1 was also expressed in 1- and 10-day-old juvenile snails whereas GHF10-Pc3 was expressed only in 1-day-old juvenile snails. The result showed that two GHF10-Pc transcripts were developmentally expressed.

Tandemly repetitive sequences are widespread in all eukaryotic genomes, but data on tandem repeats are limited in Zhikong scallop (Chlamys farreri). In the present study, paired-end sequencing of 2016 individual fosmid clones resulted in 3646 sequences. A total of 2,286,986 bp of genomic sequences were generated, representing approximately 1.84 per thousand of the Zhikong scallop genome. Using tandem repeats finder (TRF) software, a total of 2500 tandem repeats were found, including 313 satellites, 1816 minisatellites and 371 microsatellites. The cumulative length of tandem repeats was 552,558 bp, accounting for 24.16% of total length. Specifically, the length of microsatellites, minisatellites and satellites was 9425, 336,001 and 207,132 bp, accounting for 1.71, 60.81 and 37.49% of the length of tandem repeats, and 0.41, 14.69 and 9.06% of total length, respectively. The detailed information on the characteristic of all repeat units was also represented, which will provide a useful resource for physical mapping and better utilization of the existing genomic information in Zhikong scallop.

Using rapid amplification of cDNA ends (RACE), the cDNA encoding eclosion hormone (EH) was cloned from the brain of Ostrinia furnacalis. The full Osf-EH cDNA is 986 bp and contains a 267 bp open reading frame encoding an 88 amino acid preprohormone, which including a hydrophobic 26 amino acid signal peptide and a 62 amino acid mature peptide. The mature Osf-EH shows high identity with Manduca sexta (95.2%), Helicoverpa armigera (91.9%) and Bombyx mori (85.5%), but low identify with Tribolium castaneum (63.6%), Drosophila melanogaster (56.5%) and Apis mellifera (54.8%). Using the HMMSTR Prediction Server, the 3D structure of Osf-EH was modeled. There are four beta-turns and three alpha-helixes predicted in Osf-EH, with the pattern of beta-beta-alpha-alpha-beta-beta-alpha. Northern blot analysis indicated a 1.0 kb transcript present only in the brain. The Osf-EH mRNA can not be detected in other neural tissues, such as the suboesophageal ganglion, thoracic ganglion, abdominal ganglion and other non-neural tissues, such as the midgut, fat body and epidermis. The Osf-EH mRNA content in the brain was measured using the combined method of quantitative RT-PCR and Southern blotting, which reached its highest level the day before the molt.

Isopentenyl diphosphate isomerase (EC 5.3.3.2, IPI) catalyzes the revisable conversion of 5-carbon isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which are the essential precursors for isoprenoids, including anti-tumor camptothecin. Here we report cloning, characterization and functional expression of a new cDNA encoding IPI from Camptotheca acuminata. The full-length cDNA was 1143 bp long designated as CaIPI (GenBank Accession Number: DQ839416), containing an open reading frame (ORF) of 930bp which encodes a polypeptide of 309 amino acids. Bioinformatic analysis showed the cDNA sequence of CaIPI was highly homologous with other IPI gene and the deduced amino acid sequence of CaIPI was similar to known plant IPIs and contained Cys-149 and Glu-212 active sites. Phylogenic analysis indicated that all IPIs could be divided into five groups and CaIPI belonged to plant IPIs' family. The tissue expression profile analysis was carried out to investigate the transcriptional level of CaIPI in different tissues. The result showed that CaIPI expression could be detected in roots, stems and tender leaves but could not in mature leaves and fruits, and the expression levels was much higher in stems than in roots and tender leaves. Finally, CaIPI was functionally expressed in engineered Escherichia coli in which the carotenoid pathway was reconstructed. In engineered E. coli, CaIPI could facilitate the metabolic flux to the carotenoids biosynthesis and made the bacteria produce the orange beta-carotene. These confirmed that CaIPI had the typically function of IPI gene. In summary, cloning, characterization and functional expression of CaIPI will facilitate to understand the function of CaIPI at the level of molecular genetics and unveil the biosynthetic mechanism of camptothecin precursors.

Complementary DNA and genomic sequences representing two different beta-actins were isolated from a threatened freshwater fish species Hemibarbus mylodon. The beta-actin 1 and 2 encoded an identical number of amino acids (375 aa), and shared 88.8 and 99.7% of identity at coding nucleotide and amino acid levels, respectively. Genomic open reading frame (ORF) sequences of both isoforms contained five translated exons interrupted by four introns with conserved GT/AG exon/intron boundary rule. Semi-quantitative RT-PCR showed that the two isoform mRNAs were ubiquitously detected in all tissues tested, but transcript levels were variable across tissues. Phylogenetic analysis showed that H. mylodon beta-actin 1 and 2 were clustered into two distinct major and minor branches of Cypriniformes, respectively. Comparisons of the 5'-upstream region and 3'-UTR of H. mylodon beta-actin 1 also showed a high degree of homology with those of the major teleost beta-actins and warmblooded vertebrate beta-cytoskeletal actins, suggesting their more recent common origin.