OBJECTIVETo identify whether GC-box (-348 to -338) in human insulin gene promoter is a key cis-acting element.

METHODSHuman insulin gene promoter was sub-cloned into secreted alkaline phosphatase (SEAP) reporter plasmid. The deletion and mutation of GC-box in insulin gene promoter was performed. The activity of human insulin gene promoter was determined by evaluating the activity of SEAP in the supernatant of cell culture after the reporter plasmids were transfected in beta cell line betaTC3.

RESULTDeletion and mutation of GC box in human insulin gene promoter did not result in significant changes of the activity of the promoter in betaTC3.

CONCLUSIONThe GC-box is not a key cis-acting element in human insulin gene promoter.

Alkaline Phosphatase ; genetics ; metabolism ; Base Sequence ; Cell Line ; Enhancer Elements, Genetic ; GC Rich Sequence ; Gene Expression Regulation ; Humans ; Insulin ; genetics ; Promoter Regions, Genetic ; Recombinant Fusion Proteins ; genetics ; metabolism ; Sequence Deletion ; Transcription, Genetic ; Transfection

Enhancers activate transcription in a distance-, orientation-, and position-independent manner, which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes, especially at the 5' untranslated regions (5'UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40% of identified enhancers carried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks, H3K4me3 and/or H3K27ac, which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes, which overlap poorly with STARR-seq enhancers. In summary, we quantitatively identified enhancers by functional analysis in the genome of rice, an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.
Acetylation ; Base Sequence ; Deoxyribonuclease I ; metabolism ; Enhancer Elements, Genetic ; Epigenesis, Genetic ; Genes, Plant ; Genomics ; methods ; Histone Code ; genetics ; Histones ; metabolism ; Models, Genetic ; Oryza ; genetics ; Promoter Regions, Genetic ; genetics ; Repetitive Sequences, Nucleic Acid ; genetics ; Sequence Analysis, DNA ; Transcription, Genetic

The energy of interaction between complementary nucleotides in promoter sequences of E. coli was calculated and visualized. The graphic method for presentation of energy properties of promoter sequences was elaborated on. Data obtained indicated that energy distribution through the length of promoter sequence results in picture with minima at -35, -8 and +7 regions corresponding to areas with elevated AT (adenine-thymine) content. The most important difference from the random sequences area is related to -8. Four promoter groups and their energy properties were revealed. The promoters with minimal and maximal energy of interaction between complementary nucleotides have low strengths, the strongest promoters correspond to promoter clusters characterized by intermediate energy values.
AT Rich Sequence ; genetics ; Base Sequence ; Chromosome Mapping ; methods ; DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA ; methods ; Transcription, Genetic ; genetics

OBJECTIVETo finish the work of sequencing the full sequence of intron 51 of dystrophin gene and understand its characteristic of sequence.

METHODSThe whole intron 51 was sequenced by primer walking. The sequencing results were analyzed by repeat sequences, matrix attachment region (MAR) and topoisomerase II cleavage sites. The residue sequences, after removal of the repetitive sequences, were subjected to the analysis of CpG islands, promoter, open reading frame (ORF) and unidentified low copy repeat sequence.

RESULTSThe acquired intron 51 sequence was composed of 38725 bp. Repetitive sequences constituted 37.53% of total intron sequence. The overall G+C content of intron 51 was 36.34%. There are four potential MARs in intron 51. Three of them are clustered in the 12 kb region near exon 51. Numerous ORFs were found on both strands, but no homologues proteins were found in Genbank CDS transcriptional peptide, PDB, SwissProt, PIR and PRF databases.

CONCLUSIONThe expansion of intron 7 over the last 120 million years was mainly the result of L1 insertion into intron 7, and not all of repetitive sequences are associated with chromosomal rearrangement. No sequence of functional significance was found in intron 51. The results suggest that the cluster of MARs may be associated with the instability of intron 51.

Base Sequence ; CpG Islands ; genetics ; Databases, Nucleic Acid ; Dystrophin ; genetics ; Gene Deletion ; Genes ; Humans ; Introns ; genetics ; Long Interspersed Nucleotide Elements ; genetics ; Mutagenesis, Insertional ; Open Reading Frames ; Promoter Regions, Genetic ; Repetitive Sequences, Nucleic Acid ; genetics ; Sequence Analysis, DNA ; methods

Vertebrate genomes are characterized with CpG deficiency, particularly for GC-poor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5' T to 5' A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5' Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected (obs/exp) values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes (except sea urchin) but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5' T and 5' A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation. CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.
AT Rich Sequence ; Animals ; CpG Islands ; genetics ; DNA Methylation ; GC Rich Sequence ; Gene Frequency ; Genome ; Genomics ; methods ; Humans ; Invertebrates ; genetics ; Isochores ; genetics ; Mutation ; Vertebrates ; genetics

The allele frequency and mutation rate in a Short Tandem Repeat locus, Y27 were studied in 247 unrelated Korean males using polymerase chain reaction followed by high-resolution polyacrylamide gel electrophoresis, a procedure called the amplification fragment length polymorphism technique. Six alleles were noted ranging from 190 bp to 210 bp. They existed as discrete bands with 4 bp discrepancy. Among which DY3(198 bp), DY4(202 bp) were common with the frequencies of 0.408, 0.356 respectively. Other alleles, DY1(190 bp, frequency 0.020), DY2(194 bp, frequency 0.121), DY5(206 bp, frequency 0.089), DY6(210 bp, frequency 0.004) were relatively uncommon. In a 78 subject father-son study with parenthood confirmed through other genetic studies, no case of mutation was noted. As the allele number was not as large as 6 and two alleles were dominant, the discrimination power in routine individual identification was thought to be low. But in selective cases such as father-son determination or sex determination, this locus could be a valuable genetic marker and we thought these results to be common for the Korean population. These results were also compared with that of other race.
*Alleles ; Base Sequence ; DNA ; Forensic Medicine ; *Gene Frequency ; Human ; Korea ; Male ; Molecular Sequence Data ; *Repetitive Sequences, Nucleic Acid ; Support, Non-U.S. Gov't

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.
Base Sequence ; Dermatoglyphics ; Genome ; Global Health ; Methods* ; Molecular Typing ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Repetitive Sequences, Nucleic Acid ; Tandem Repeat Sequences ; Tuberculosis

Southern blot analysis with probe from mini-Tn5 gfp-km transposon indicated that 5 non-pathogenic mutants which were generated by insertion of mini-Tn5 gfp-km mutagenesis contained a single copy of the transposon. Using genomic DNA of each mutant as a template, TAIL-PCR was performed with seven arbitrary degenerate (AD) primers pairing with 3 nested specific primers designed based on the sequence of GFP toward outside in mini-Tn5 gfp-km. After 3-step PCR reactions, the flanking sequence of each mutant was obtained. The PCR product was ligated with pGEM-T EASY vector and then was transformed into E. coli DH5 alpha by electroporation. Positive clones were selected by white/blue colony and plasmid was isolated, then digested with EcoRI. Plasmid was sequenced if its insert was longer than 300 bp. Our results indicated that TAIL-PCR was proved to be a simple and efficient approach in identification of gene using insertion mutagenesis.
Base Sequence ; Cloning, Molecular ; methods ; DNA Transposable Elements ; DNA, Bacterial ; isolation & purification ; Genes, Bacterial ; Green Fluorescent Proteins ; Luminescent Proteins ; genetics ; Molecular Sequence Data ; Mutagenesis ; Polymerase Chain Reaction ; methods ; Xanthomonas campestris ; genetics ; pathogenicity

The DNA sequence of a plasmid named pHP489 of Helicobacter pylori strain 489 was determined and analyzed to characterize its replication apparatus. The pHP489 plasmid consisted of 1,222 bp and had an overall G+C content of 33.1%. An ORF was predicted to encode the putative protein of 239 amino acid residues (28 kDa). A putative ribosomal binding site and a potential terminator sequence are located upstream and downstream of the ORF, respectively. However, the consensus sequence for a promoter in upstream of ORF was not found. A potential dna A box was found at 317 nt upstream of a start codon and followed by two-57 bp directed repeats and an inverted repeat. The DNA homology was found in the regions of less than 90 bp among pHPK255, pHPM180, and pHel1 of other H. pylori plasmids and Mycoplasma mycoides plasmids. pHP489K that was produced by pHP489 sequence and C. jejuni derived aph(3')-III, was transformed to various H. pylori isolates and were stably maintained in the H. pylori host without the addition of selective antibiotics for the 30-times subcultues. The plasmic vector, in which the ORF region of pHP489 DNA was deleted, could be transformed into H. pylori. However, the plasmid vector, whose the direct repeats region of pHP489 DNA was deleted, failed to be transformed. The direct repeats region of pHP489 DNA was confirmed to be bound with cytosolic factors of H. pylori. These results showed that the direct repeats region of pHP489 DNA is an essential apparatus by which the plasmid could be replicacted in H. pylori. And pHP489 plasmid was supposed to be replicated by host factors rather than plasmic-encoded factors.
Animals ; Anti-Bacterial Agents ; Base Composition ; Base Sequence ; Binding Sites ; Codon, Initiator ; Consensus Sequence ; Cytosol ; DNA ; Ecthyma, Contagious ; Helicobacter pylori* ; Helicobacter* ; Mycoplasma mycoides ; Plasmids* ; Repetitive Sequences, Nucleic Acid ; Sequence Analysis* ; Terminator Regions, Genetic

No abstract available.
Base Sequence*