Continuous, on‐line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus

Middendorf, Lyle R.; Bruce, J.C.; Bruce, Robert; Eckles, Robert D.; Grone, Daniel L.; Roemer, Stephen C.; Sloniker, Gary D.; Steffens, David L.; Sutter, Scott L.; Brumbaugh, J. A.; Patonay, Gábor

doi:10.1002/elps.11501301103

Cited by 162 publications

(68 citation statements)

References 24 publications

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“…Complete coding information for the Giardia calmodulin ORF was obtained by directed sequencing of EJ2820. We amplified the clone insert using PCR and primers specific to the Giardia sequences, and then sequenced this product completely using a LICOR automated scanner (27). The sequence has since been confirmed by additional random sequence data from the genome project data base and by sequencing constructs.…”

Section: Methodsmentioning

confidence: 99%

Calcium Signaling in Excystation of the Early Diverging Eukaryote, Giardia lamblia

Reiner

Hetsko

Meszaros

et al. 2003

Journal of Biological Chemistry

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Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic differentiation induced by physiological signals from the host. Our data implicate a central role for calcium homeostasis in excystation. Agents that alter cytosolic Ca 2؉ levels (1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acidtetra(acetyloxymethyl) ester, a Ca 2؉ channel blocker, Ca 2؉ ionophores, and thapsigargin) strongly inhibit excystation. Treatment of Giardia with thapsigargin raised intracellular Ca 2؉ levels, and peak Ca 2؉ responses increased with each stage of excystation, consistent with the kinetics of inhibition. Fluorescent thapsigargin localized to a likely Ca 2؉ storage compartment in cysts. The ability to sequester ions in membranebounded compartments is a hallmark of the eukaryotic cell. These studies support the existence of a giardial thapsigargin-sensitive Ca 2؉ storage compartment resembling the sarcoplasmic/endoplasmic reticulum calcium ATPase pump-leak system and suggest that it is important in regulation of differentiation and appeared early in the evolution of eukaryotic cells. Calmodulin antagonists also blocked excystation. The divergent giardial calmodulin localized to the eight flagellar basal bodies/centrosomes, like protein kinase A. Inhibitor kinetics suggest that protein kinase A signaling triggers excystation, whereas calcium signaling is mainly required later, for parasite activation and emergence.

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Section: Methodsmentioning

confidence: 99%

Calcium Signaling in Excystation of the Early Diverging Eukaryote, Giardia lamblia

Reiner

Hetsko

Meszaros

et al. 2003

Journal of Biological Chemistry

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“…Double-stranded sequencing of the rRNA coding regions was accomplished using either Sequenase Ver. 2.0 (United States Biochemical) and existing rDNA primers (20,21) or using a Li-Cor 4000L automated sequencer and Sequitherm cycle sequencing protocols developed by Li-Cor (Lincoln, NE) (22).…”

Section: Methodsmentioning

confidence: 99%

Phylogenetic relationships between the Acantharea and the Polycystinea: A molecular perspective on Haeckel’s Radiolaria

Zettler

Sogin

Caron

1997

Proc. Natl. Acad. Sci. U.S.A.

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Polycystine radiolaria are among few protistan groups that possess a comprehensive fossil record available for study by micropaleontologists. The Polycystinea and the Acantharea, whose skeletons do not become fossilized, were once members of the class ''Radiolaria'' (''Radiolaria'' sensu lato: Polycystinea, Phaeodarea, and Acantharea) originally proposed by Haeckel but are now included in the superclass Actinopoda. Phylogenetic relationships within this superclass remain largely enigmatic. We investigated the evolutionary relationship of the Acantharea and the Polycystinea to other protists using phylogenetic analyses of 16S-like ribosomal RNA (rRNA) coding regions. We circumvented the need to culture these organisms by collecting and maintaining reproductive stages that contain many copies of their genomic DNA. This strategy facilitated extraction of genomic DNA and its purification from symbiont and prey DNA. Phylogenetic trees inferred from comparisons of 16S-like coding regions do not support a shared history between the Acantharea and the Polycystinea. However, the monophyly of the Acantharea and the separate monophyly of the Polycystinea (Spumellarida) are well supported by our molecularbased trees. The acantharian lineage branches among crown organisms whereas the polycystine lineage diverges before the radiation of the crown groups. We conclude that the Actinopoda does not represent a monophyletic evolutionary assemblage and recommend that this taxonomic designation be discarded.

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“…Sequenced PCR products were run on polyacrylamide gels made with Sequagel XR (National Diagnostics, Hessle, UK) using the Li-Cor Global IR2 System, which allows simultaneous bidirectional sequencing from one reaction. 21 SNPs selected for screening were genotyped by KBiosciences (Hertfordshire, UK) which employs either the Amplifluor, KASPar or Taqman SNP genotyping methods. Internal controls were used to check for accuracy with approximately 17% of samples duplicated in order to detect error and confirm the reproducibility of genotypes.…”

Section: Clinical Samplingmentioning

confidence: 99%

A threonine to isoleucine missense mutation in the pericentriolar material 1 gene is strongly associated with schizophrenia

et al. 2008

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Markers at the pericentriolar material 1 gene (PCM1) have shown genetic association with schizophrenia in both a University College London (UCL) and a USA-based case-control sample. In this paper we report a statistically significant replication of the PCM1 association in a large Scottish case-control sample from Aberdeen. Resequencing of the genomic DNA from research volunteers who had inherited haplotypes associated with schizophrenia showed a threonine to isoleucine missense mutation in exon 24 which was likely to change the structure and function of PCM1 (rs370429). This mutation was found only as a heterozygote in 98 schizophrenic research subjects and controls out of 2246 case and control research subjects. Among the 98 carriers of rs370429, 67 were affected with schizophrenia. The same alleles and haplotypes were associated with schizophrenia in both the London and Aberdeen samples. Another potential aetiological base pair change in PCM1 was rs445422, which altered a splice site signal. A further mutation, rs208747, was shown by electrophoretic mobility shift assays to create or destroy a promoter transcription factor site. Five further non-synonymous changes in exons were also found. Genotyping of the new variants discovered in the UCL case-control sample strengthened the evidence for allelic and haplotypic association (P = 0.02-0.0002). Given the number and identity of the haplotypes associated with schizophrenia, further aetiological base pair changes must exist within and around the PCM1 gene. PCM1 protein has been shown to interact directly with the disrupted-in-schizophrenia 1 (DISC1) protein, BardetBiedl syndrome 4, and Huntingtin-associated protein 1, and is important in neuronal cell growth. In a separate study we found that clozapine but not haloperidol downregulated PCM1 expression in the mouse brain. We hypothesize that mutant PCM1 may be responsible for causing a subtype of schizophrenia through abnormal cell division and abnormal regeneration in dividing cells in the central nervous system. This is supported by our previous finding of orbitofrontal volumetric deficits in PCM1-associated schizophrenia patients as opposed to temporal pole deficits in non-PCM1-associated schizophrenia patients. Caution needs to be exercised in interpreting the actual biological effects of the mutations we have found without further cell biology. However, the DNA changes we have found deserve widespread genotyping in multiple case-control populations.

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Continuous, on‐line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus

Cited by 162 publications

References 24 publications

Calcium Signaling in Excystation of the Early Diverging Eukaryote, Giardia lamblia

Calcium Signaling in Excystation of the Early Diverging Eukaryote, Giardia lamblia

Phylogenetic relationships between the Acantharea and the Polycystinea: A molecular perspective on Haeckel’s Radiolaria

A threonine to isoleucine missense mutation in the pericentriolar material 1 gene is strongly associated with schizophrenia

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