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At least 811 records · Page 45Linked to original sources
PubMed2003

Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR.

The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system. It shows 100% gene identity to homologous gene in Pyrococcus furiosus. The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E. coli BL21(DE3)pLysS and E. coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E. coli tRNAs. E. coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E. coli BL21(DE3)pLysS. The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated. We found that His(6)-tag domain has no influence on dUTP hydrolytic activity. dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity. We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P. woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets.

Archaeal Proteins↗
PubMed2003

Ancient DNA analysis reveals woolly rhino evolutionary relationships.

With ancient DNA technology, DNA sequences have been added to the list of characters available to infer the phyletic position of extinct species in evolutionary trees. We have sequenced the entire 12S rRNA and partial cytochrome b (cyt b) genes of one 60-70,000-year-old sample, and partial 12S rRNA and cyt b sequences of two 40-45,000-year-old samples of the extinct woolly rhinoceros (Coelodonta antiquitatis). Based on these two mitochondrial markers, phylogenetic analyses show that C. antiquitatis is most closely related to one of the three extant Asian rhinoceros species, Dicerorhinus sumatrensis. Calculations based on a molecular clock suggest that the lineage leading to C. antiquitatis and D. sumatrensis diverged in the Oligocene, 21-26 MYA. Both results agree with morphological models deduced from palaeontological data. Nuclear inserts of mitochondrial DNA were identified in the ancient specimens. These data should encourage the use of nuclear DNA in future ancient DNA studies. It also further establishes that the degraded nature of ancient DNA does not completely protect ancient DNA studies based on mitochondrial data from the problems associated with nuclear inserts.

Animals↗
PubMed2003

Molecular genetic identification of crustose representatives of the order Corallinales (Rhodophyta) in Chile.

Knowledge on species of the order Corallinales along the coast of Chile is still scarce despite a number of studies and records of other divisions of seaweeds made since the early 20th century. This lack of information is more dramatic among crustose representatives of the order, thus depriving biogeographic studies of a thorough analysis and resulting in inadequately representative accounts of biodiversity. The currently changing taxonomy of the group makes it difficult to identify and differentiate among taxa based on morphological and developmental characters. Therefore, the use of molecular tools has been adopted in this study in order to facilitate identification and comparison of crustose corallines collected at the rocky intertidal between 27 degrees and 48 degrees S along the Pacific temperate coast of South America. A sequence 600bp (in length) from the SSU-rDNA gene was used to identify five taxa to the genus level: Lithophyllum, Spongites, Mesophyllum, Synarthrophyton, and Leptophytum. In all cases, the genus distinction based on morphological characters coincide with designations based on variation in the ribosomal DNA gene sequence. Spongites is the most frequently occurring genus and is found in all localities sampled while the others appear occasionally. Taxa recognition at species level must be examined with caution considering that morphological variability is not well understood in Chile because the SSU-rDNA region sequence does not always stand alone as an unambiguous means of identifying all coralline species. In such cases, more rapidly evolving markers are needed. For example, sequences from the ITS (rDNA) region often provide greater resolution among closely related species and genera. However, the methodology presented here remains a useful tool for species-level identification.

Animals↗
PubMed2003

Spectrum of mutations in the arylsulfatase A gene in a Canadian DNA collection including two novel frameshift mutations, a new missense mutation (C488R) and an MLD mutation (R84Q) in cis with a pseudodeficiency allele.

We describe three novel mutations in the human arylsulfatase A gene in three patients with MLD, an autosomal recessive lysosomal storage disorder. An insertion, 2590_2591insCCCC in exon 8 and a deletion, 752_758delGCCGGCC, in exon 3 will both result in frameshifts. A mutation in exon 8, 2566T-->C, results in a missense mutation C488R, disrupting an unusual cysteine-knot at the C-terminal end of the protein. All three mutations are heterozygous with previously documented mutations. A previously reported mutation, R84Q was identified on a pseudodeficiency allele. These mutations are part of a heterogeneous spectrum of mutations found in a collection of DNA samples from MLD patients from across Canada and the USA.

Adolescent↗
PubMed2002

Section 5: Structural/genetic analysis of mAbs to blood group antigens. Coordinator's report.

The heavy and light chain immunoglobulin variable region nucleotide sequences for 219 mAbs to human red blood cells were collected from workshop participants, published reports, and Genbank. Information regarding antigen specificity, species of origin, method of cloning, and other relevant serological properties was correlated with the sequence data. Immunoglobulin sequences were analyzed to determine the heavy- and light-chain immunoglobulin genes used and the overall extent of somatic mutation from germline configuration. Approximately 50% of the sequences encoded antibodies with Rh(D) specificity with the remaining sequences encoding mAbs to other Rh-related antigens, antigens of the ABO, MNS, and Kell blood group systems, and several others. Surprisingly, no sequence data were available for mAbs with specificity for a number of common Rh antigens, common Kell antigens, or antigens of the Lewis, Kidd, or Duffy blood group systems. The majority of mAbs were of human origin but included a significant number of macaque mAbs, murine mAbs, and a small number of synthetically-designed recombinant antibodies. Both cellular (EBV-transformation, cell fusion) and molecular (phage display) approaches were used for antibody cloning. Analysis of certain groups of sequences demonstrated patterns of immunoglobulin gene restriction, repertoire shift, and somatic mutation. Analysis of other mAbs demonstrated the value of antibody sequence data for the design and production of novel reagents useful in blood group serology.

Animals↗
PubMedDate not supplied

The collation of forensic DNA case data into a multi-dimensional intelligence database.

The primary aim of any DNA Database is to link individuals to unsolved offenses and unsolved offenses to each other via DNA profiling. This aim has been successfully realised during the operation of the New Zealand (NZ) DNA Databank over the past five years. The DNA Intelligence Project (DIP), a collaborative project involving NZ forensic and law enforcement agencies, interrogated the forensic case data held on the NZ DNA databank and collated it into a functional intelligence database. This database has been used to identify significant trends which direct Police and forensic personnel towards the most appropriate use of DNA technology. Intelligence is being provided in areas such as the level of usage of DNA techniques in criminal investigation, the relative success of crime scene samples and the geographical distribution of crimes. The DIP has broadened the dimensions of the information offered through the NZ DNA Databank and has furthered the understanding and investigative capability of both Police and forensic scientists. The outcomes of this research fit soundly with the current policies of 'intelligence led policing', which are being adopted by Police jurisdictions locally and overseas.

Databases, Nucleic Acid↗
PubMed1998

The Human Genome Diversity Project: medical benefits versus ethical concerns.

By the year 2005 the entire human genome should have been sequenced and the genes identified. But the resulting genomic sequence, although a marvelous accomplishment, will be a composite of just a handful of individuals selected at random. The Human Genome Diversity Project was proposed as a means to overcome these limitations by obtaining genetic information from many diverse populations of the world. This would give medical geneticists a handle on the variations in susceptibility to disease among different populations, as well as being of anthropological value. But would such a project risk exploiting the indigenous populations involved?

Bioethics↗
PubMed2002

From microarrays to networks: mining expression time series.

Over the past few years, powerful new methods have been devised that enable researchers to study the expression dynamics of many genes simultaneously (e.g. gene expression profiles using cDNA microarrays). In principle, this potentially vast quantity of data enables the dissection of the complex genetic networks that control the patterns and rhythms of gene expression in the cell. Finding the patterns in those data represents the next major phase in our understanding of the programming and functioning of the living cell. Simple dynamic models can be used to generate gene expression networks. These networks reveal the phenomenological link between the expression of different genes. This review discuss how these networks are generated and outlines several data-mining techniques for extracting relationships and hypotheses in gene expression. These emerging methods can be applied to a range of biological problems.

Computational Biology↗
PubMed2002

Rational drug discovery: what can we learn from regulatory networks?

To enable the list of genes and proteins contained within genomic databases to be useful for drug discovery, we need to understand how the genome maps into the phenome. An essential, but not explicitly listed ingredient of the genome is the regulatory interactions between genes and proteins that form a genome-wide network. How can the concept of regulatory networks increase our understanding of living systems? Networks are more than just static "wiring diagrams". Gene interactions impose dynamic constraints, which, although obvious in emergent phenotypic properties, are not captured by traditional one gene-one trait approaches. Understanding the nature of these constraints in gene-activation state space will pave the way to a holistic yet formal and genomics-based approach to rational drug development.

Computational Biology↗
PubMed2002

Human gene discovery through experimental definition of transcribed regions of the human genome.

The sequencing of the human genome has failed to realize its primary goal: the identification of all human genes. We have learned that genes can only be identified with certainty within this vast and information-sparse structure by comparison with transcript sequences. Significantly more sequence data of this kind is required before we can claim to have deciphered our genetic blueprint.

Algorithms↗
PubMed2002

Staking out novelty on the genomic frontier.

Recent advances in genomics include global assessment and classification of genome content, high-throughput biological pathway construction, systematic identification of previously unpredicted genes and the in vitro creation of novel motifs with biological function not found in nature (extra-genomic gene discovery). The ability to make global surveys of transcriptomes has given rise to fields such as pharmacogenomics and toxicogenomics. These applications of genomics technologies, with conventional drug assessment methodologies, will lead to more tolerable drugs and a better understanding of clinical populations. Integration of pathway mapping, using proteomics married to expression, will also significantly affect how new therapeutics are discovered as cross-biological cross-pathway interactions lead to novel drug targets and better predictions of drug tolerance.

Alternative Splicing↗
PubMed2002

How can we deliver the large plant genomes? Strategies and perspectives.

The first sequenced plant genome, from the small mustard plant Arabidopsis thaliana, was published at the end of 2000. The sequencing of the rice genome is well under way. The sizes of plant genomes vary by a factor of up to 1000, and many important crop plants have genomes that are several times larger than the human genome. To gain insight into the gene toolbox of plant species, numerous large-scale EST sequencing projects have been launched successfully, and analysis procedures are constantly being refined to add maximum value to the sequence data. In addition, an alternative approach to exclude repetitive noncoding DNA and to enrich sequence libraries for gene-containing genomic regions has been developed. This strategy has the potential to deliver information about both genes and regulatory regions outside the transcribed regions.

Arabidopsis↗