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PubMed1986

Consecutive DNA terminator sequencing by using enzymatically generated primers.

An improved strategy for the dideoxy chain termination sequencing of M13 recombinant DNA using enzymatically generated primers is presented. It involves synchronous extension of the universal primer with the Klenow fragment of DNA polymerase I at an average rate of 200 deoxynucleoside triphosphates per minute to the immediate downstream region of a preselected restriction enzyme site. Subsequent cleavage with the restriction enzyme generates the short primers with homogeneous 5' ends which can be used for further sequencing. The next restriction sites are selected in the newly sequenced regions of DNA by means of a microcomputer. By repeating this primer extension-cleavage-sequencing strategy sequences altogether about 6 kb long from several recombinant single-stranded M13 DNAs have been determined by using twenty restriction enzymes with different specificities.

Base Sequence↗

PubMed1996

Evolving cooperation: the role of individual recognition.

To evaluate the role of individual recognition in the evolution of cooperation, we formulated and analyzed genetic algorithm model (EvCo) for playing the Iterated Prisoner's Dilemma (IPD) game. Strategies compete against each other during each generation, and successful strategies contribute more of their attributes to the next generation. Each strategy is encoded on a 'chromosome' that plays the IPD, responding to the sequences of most recent responses by the interacting individuals (chromosomes). The analysis reported in this paper considered different memory capabilities (one to five previous interactions), pairing continuities (pairs of individuals remain together for about one, two, five, or 1000 consecutive interactions), and types of individual recognition (recognition capability was maximal, nil, or allowed to evolve between these limits). Analysis of the results focused on the frequency of mutual cooperation in pairwise interactions (a good indicator of overall success in the IPD) and on the extent to which previous responses by the focal individual and its partner were associated with the partner's identity (individual recognition). Results indicated that a fixed, substantial amount of individual recognition could maintain high levels of mutual cooperation even at low pairing continuities, and a significant but limited capability for individual recognition evolved under selection. Recognition generally increased mutual cooperation more when the recent responses of individuals other than the current partner were ignored. Titrating recognition memory under selection using a fitness cost suggested that memory of the partner's previous responses was more valuable than memory of the focal's previous responses. The dynamics produced to date by EvCo are a step toward understanding the evolution of social networks, for which additional benefits associated with group interactions must be incorporated.

Algorithms↗

PubMed2004

[Sequencing and analyzing of expressed sequence tags (ESTs) of pig fat tissue].

In this paper,the ESTs of pig fat tissue were sequenced and analyzed using the large-scale DNA sequencing method, 7790 high quality ESTs were gained,from which 4354 genes were obtained using cluster analysis with the STACK-PACK software, including 3609 singlet genes and 745 multicopy genes. The 4354 candidate genes were compared with BLASTN to the nr library (e =1e-10), of which 2712 were known genes, containing 1 987 singlet genes and 725 multicopy genes,there are 2 109 unknown genes and new ESTs. Based on the results of BlastN and the index of GenBank Accenssion No., the known gene expression profile of pig fat were constructed. It showed that the genes participating in metabolism held the highest proportion in the 7 sorts, and in some aspects showed the hearty metabolism activity of fat tissue. 257 total reads of the Major Histocompatibility Complex (MHC) genes in the pig fat tissue were found,including 181 singlet genes and 44 multicopy genes. They account for 44.9% of the cell and organism defense, and 5.4% of all known genes,from this we can see that the MHC has a fairly high expression degree. Comparing all ESTs related with MHC (257reads) with known pig BAC we found the partial sequences (approximately 200 bp) of the ESTs distributing to every exon of. We can predict that the ESTs in MHC contain approximately 200 bp highly consistent regions and every exon possesses one of these regions. Although these MHC sequences in different BAC have different protein domains,they are highly consistent and related with immune functions. When the MHC genes transfer to the next generation,they can duplicate repeatly and inherit stably.

Adipose Tissue↗

PubMed2002

Molecular diagnosis of HIV.

The development of molecular techniques that access viral load and the development of genotypic resistance have revolutionized the treatment of HIV disease. Commercially available viral load assays use a number of different approaches from reverse transcriptase PCR to amplification of branched chain DNA. The drawbacks of the assay are that there is no international standard that allows comparison of viral load between assays and the diversity of different clades of HIV results in the under or the nondetection of some patients samples. New real-time PCR assays are under development, including LightCycler- and TaqMan-based tests. The development of sequence-based genotyping assays for the detection of mutations associated with the development of the resistance to the 17 licensed drugs targeted against the pol gene of HIV have added to the improvements in patient management. However, next-generation assays must extend detection to include the gp41 fusion region and the integrase region of the genome as compounds directed against these targets move from clinical trails into licensed drugs. Also, genotypic assays must improve detection of minor species and detection of sequences from patients with low viral load number. Real-time sequence based-diagnostics remains a realistic target within the next 5 years.

Anti-HIV Agents↗

PubMed2006

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering.

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.

Chemical Engineering↗

PubMed1987

Characterization of a highly polymorphic region 5' to JH in the human immunoglobulin heavy chain.

A cloned DNA segment 1.25 kilobases (kb) upstream from the joining segments of the human heavy chain immunoglobulin gene revealed extensive polymorphic variation at this locus, and the polymorphic pattern was stably transmitted to the next generation. Genomic restriction analysis showed that the polymorphism was caused by insertions/deletions within an MspI/BamHI fragment. Sequencing of one allele, 848 base pairs (bp) long, revealed eleven 50-base-pair tandem repeats. A second allele, 648 bp long, was cloned from a human genomic cosmid library, sequenced, and found to contain four fewer repeats than the first allele. A survey of 186 chromosomes from unrelated individuals of primarily northern European descent revealed at least six alleles.

Alleles↗

PubMed1985

[Transformation of mice by a prokaryotic gene for dihydrofolate reductase].

In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found. About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e. are transgenic. In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR. The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice.

Animals↗

PubMed1996

A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation.

CTL epitope (KSPWFTTL) encoded by AKV/MCF type of murine leukemia virus (MuLV) differs from the sequence in Friend/Moloney/Rauscher (FMR) type in one residue (RSPWFTTL). CTL experiments indicated defective processing of the FMR peptide in tumor cells. Proteasome-mediated digestion of AKV/MCF-type 26-mer peptides resulted in the early generation and higher levels of epitope-containing fragments than digestion of FMR-type peptides, explained by prominent cleavage next to R in the FMR sequence. The fragments were identified as 10- and 11-mer peptides and were efficiently translocated by TAP. The naturally presented AKV/MCF peptide is the 8-mer, indicating ER peptide trimming. In conclusion, a single residue exchange can cause CTL epitope destruction by specific proteasomal cleavage.

ATP-Binding Cassette Transporters↗

PubMed1989

Novel third-letter bias in Escherichia coli codons revealed by rigorous treatment of coding constraints.

A novel bias in codon third-letter usage was found in Escherichia coli genes with low fractions of "optimal codons", by comparing intact sequences with control random sequences. Third-letter usage has been found to be biased according to preference in codon usage and to doublet preference from the following first letter. The present study examines third-letter usage in the context of the nucleotide sequence when these preferences are considered. In order to exclude any influence by these factors, the random sequences were generated such that the amino acid sequence, codon usage, and the doublet frequency in each gene were all preserved. Comparison of intact sequences with these randomly generated sequences reveals that third letters of codons show a strong preference for the purine/pyrimidine pattern of the next codons: purine (R) is preferred to pyrimidine (Y) at the third site when followed by an R-Y-R codon, and pyrimidine is preferred when followed by an R-R-Y, an R-Y-Y or a Y-R-Y codon. This bias is probably related to interactions of tRNA molecules in the ribosome.

Amino Acids↗

PubMed1998

Molecular cloning of a light-inducible gene (lipA) encoding a novel pilin from Arthrobacter photogonimos.

Development of pili on cells of Arthrobacter photogonimos is induced by photo-oxidative conditions. The nucleotide sequence was determined of a light-inducible gene (lipA) that encodes the precursor of a light-inducible pilin (designated LIP), a polypeptide of 212 amino acids. The N-terminal leader peptide includes a typical signal sequence with a consensus cleavage site for signal peptidase 1 after residue 28, which should generate N-terminal arginine. However, the next amino acid, alanine, is the N-terminal residue of the mature protein. The abundance of charged amino acids (27% of total), a calculated pI of 9.98, and recovery of mostly monomers when cells were washed with 1 M NaCl suggest that electrostatic interactions play a dominant role in association of LIP, a novel mechanism for assembly of pili.

Amino Acid Sequence↗

PubMed2000

Function of the sigma(E) regulon in dead-cell lysis in stationary-phase Escherichia coli.

Elevation of active sigma(E) levels in Escherichia coli by either repressing the expression of rseA encoding an anti-sigma(E) factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase E. coli occurs in a sigma(E)-dependent fashion. We propose there is a novel physiological function of the sigma(E) regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.

Amino Acid Sequence↗

PubMed1998

Adeno-associated virus vector-mediated transgene integration into neurons and other nondividing cell targets.

The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.

Animals↗

PubMed2000

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.

Actins↗

PubMed2001

Statistics of the integrated backscatter estimate from a blood-mimicking fluid.

This work evaluates the variance of the integrated backscatter (IBS) from moving blood [or blood-mimicking fluid (bmf)] as a way of determining the quality of the mean IBS estimate. The main motivation for this work comes from the fact that absolute IBS values from tissues adjacent to arterial blood can be found by normalizing the measured backscatter energy with the IBS of moving, deaggregated blood. The paper describes the parameters that control the statistics of the IBS estimate, which is calculated for the stochastic ultrasound backscatter signals from flowing blood. It further formulates how the measurement parameters should be specified so that an appropriately low blood IBS variance is ensured or, alternatively, a specified accuracy of the tissue IBS estimate is obtained. First, the paper provides an analytic formulation of the statistics of the IBS, based on a sequence of sampled echoes from a nonstationary Gaussian scattering medium. The analysis incorporates the correlation between the sample values as well as the correlation between the IBS of the individual echoes. The estimate of the mean IBS has been shown to be chi-squared distributed with a determinable order. With the degree of correlation between the samples and between the IBS of individual echoes specified, the number of measurements required to obtain an IBS estimate with a specified variance is readily calculated. Next, a sequence of synthetic echoes is produced and arranged as columns in a data matrix. The echoes are generated such that the second-order statistics along the rows and columns of the matrix match that of actually observed echoes. The actual variance of the mean IBS estimate for the synthetic echoes is calculated and compared with the variance determined from the analytic model, and a good agreement has been found. Finally, sequences of actual backscattered echoes from circulating blood-mimicking fluid are acquired and analyzed to determine the variance of their mean IBS estimate. Based on the measured second-order statistics of the rows and columns of the data matrix for the actual echoes, the observed variance of the mean IBS estimate was compared with the analytically determined variance and with good agreement. Thus, the paper has shown through modeling, simulations, and experiments how the variance of the IBS estimate of the blood backscatter signal can be quantified and reduced to a specified tolerable level.

Blood↗

PubMed2006

Hypervariable region structure and polymorphism of mtDNA from dental pulp and a family analysis.

Nucleotide sequences of the hypervariable region in the D-loop of mitochondrial DNA (mtDNA) were analyzed using DNA extracted from 140 old dental pulp samples. These sequences were compared with the sequence reported by Anderson et al. Nucleotide substitution in the HV1 region was identified at 77 positions. A C-to-T transition at position 16223 (C16223T) was most frequently detected (77.9%). Fourteen types of C-stretch sequence patterns were detected and the same sequence as Anderson had the highest frequency (57.9%). In the HV2 region, base transitions were identified at 56 positions. A263G was identified in all samples. Seven types of C-stretch were detected, but none had the same sequence as Anderson. In the HV3 region, base transitions were identified at 21 positions. T489C was most frequently identified (64.3%). Five types of C-stretch were detected, and the same sequence as Anderson accounted for 92.9%. The 140 samples were classified into 128 kinds by the sequence patterns of the HV region. Next, using the blood and oral mucosa epithelium from 23 subjects comprising four generations in a family line, the hereditary relationship of mtDNA was examined. All mtDNA types of the first-generation mother were infallibly inherited by the fourth generation.

Adult↗

PubMed2001

Guide to the draft human genome.

There are a number of ways to investigate the structure, function and evolution of the human genome. These include examining the morphology of normal and abnormal chromosomes, constructing maps of genomic landmarks, following the genetic transmission of phenotypes and DNA sequence variations, and characterizing thousands of individual genes. To this list we can now add the elucidation of the genomic DNA sequence, albeit at 'working draft' accuracy. The current challenge is to weave together these disparate types of data to produce the information infrastructure needed to support the next generation of biomedical research. Here we provide an overview of the different sources of information about the human genome and how modern information technology, in particular the internet, allows us to link them together.

Amino Acid Sequence↗

PubMed1998

Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells.

To analyze genomic changes resulting from double-strand break (DSB) repair, transgenic tobacco plants were obtained that carried in their genome a restriction site of the rare cutting endonuclease I-SceI within a negative selectable marker gene. After induction of DSB repair via Agrobacterium-mediated transient expression of I-SceI, plant cells were selected that carried a loss-of-function phenotype of the marker. Surprisingly, in addition to deletions, in a number of cases repair was associated with the insertion of unique and repetitive genomic sequences into the break. Thus, DSB repair offers a mechanism for spreading different kinds of sequences into new chromosomal positions. This may have evolutionary consequences particularly for plants, as genomic alterations occurring in meristem cells can be transferred to the next generation. Moreover, transfer DNA (T-DNA), carrying the open reading frame of I-SceI, was found in several cases to be integrated into the transgenic I-SceI site. This indicates that DSB repair also represents a pathway for the integration of T-DNA into the plant genome.

Base Sequence↗

PubMed2025

Permutation tests to assess sex differences in omics data.

It is common to sex-stratify analyses of omics data and to report effects as 'sex-specific' when they are significant in only one sex. However, when analysing hundreds or thousands of molecules, this approach will yield many spurious 'sex-specific' effects if not supported by significant interactions. I illustrate this problem using an RNA sequencing dataset showing almost no significant sex by treatment interactions, but where sex-stratified analyses yield hundreds of 'sex-specific' effects of treatment. These 'sex-specific' effects could be spurious or could be real but not show interactions due to low statistical power. To distinguish these possibilities, I describe permutation tests, which provide an intuitive way to determine if a pattern of observations differs from what would be expected due to chance. For this dataset, assigning sex at random often generates more 'sex-specific' effects than the real data, demonstrating that there is little evidence of sex differences. Next, I simulate an RNA sequencing dataset that includes genes modelled to have sex-specific effects of a condition. As expected, analysis of this simulated dataset yields both significant interactions and sex-specific effects in sex-stratified analyses. While stratified analyses detect a higher number of sex-specific effects than the analysis of interactions, they erroneously identify genes not modelled to show sex-specific effects more often than interactions. A permutation test confirms that the number of sex-specific effects observed in the simulated dataset is greater than expected due to chance. Permutation tests can be applied to omics studies of sex differences, simultaneously providing (i) a clear and simple demonstration of the problems of sex-stratified analyses, and (ii) additional evidence of sex-specific effects where these are present. R code is provided for permutations, simulations, and plots to visualize potential sex-specific effects, which can be adapted to other types of data.

Female↗