Finishing the finished human chromosome 22 sequence

Background

Although the human genome sequence was declared complete in 2004, the sequence was interrupted by 341 gaps of which 308 lay in an estimated approximately 28 Mb of euchromatin. While these gaps constitute only approximately 1% of the sequence, knowledge of the full complement of human genes and regulatory elements is incomplete without their sequences.

Results

We have used a combination of conventional chromosome walking (aided by the availability of end sequences) in fosmid and bacterial artificial chromosome (BAC) libraries, whole chromosome shotgun sequencing, comparative genome analysis and long PCR to finish 8 of the 11 gaps in the initial chromosome 22 sequence. In addition, we have patched four regions of the initial sequence where the original clones were found to be deleted, or contained a deletion allele of a known gene, with a further 126 kb of new sequence. Over 1.018 Mb of new sequence has been generated to extend into and close the gaps, and we have annotated 16 new or extended gene structures and one pseudogene.

Conclusion

Thus, we have made significant progress to completing the sequence of the euchromatic regions of human chromosome 22 using a combination of detailed approaches. Our experience suggests that substantial work remains to close the outstanding gaps in the human genome sequence.     

FLAGELLAR ROOTS AND THE RESERVOIR CYTOSKELETON OF COLACIUM LIBELLAE (EUGLENOPHYCEAE)1

The three flagellar roots of Colacium Ehrenberg give rise to the three microtublar bands of the reservoir cytoskeleton. The dorsal root (DR) originates at the basal body (bb1) of the emergent flagellum. It is initiated on the left side of the cell, runs toward the right side under the posterior end of the reservoir and thence anteriorly in a spiral path over the dorsal surface of the reservoir until it terminates on the left side of the eyespot. Along its length, it appears to initiate a dorsal band (DB) which forms the major dorsal portion of the reservoir cytoskeleton—the dorsal microtubules (DMT). Two roots originate at the basal body (bb2) of the non-emergent flagellum. The ventral root (VR) runs up the left side of the cell and initiates the band of microtubules which forms part of the presumptive vestigial cytopharynx. Therefore, it forms the reinforcing microtubules (MTR) of Colacium. The intermediate root (IR) forms the para-reservoir microtubules (PMT). Flagellar root correlation with the reservoir cytoskeletal bands strengthens their homologies with the bodonid bands and further supports the hypothesis that the euglenoids are derived from the kinetoplastid flagellates.     

High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol^®), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 μm column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10–1000 ng/ml (0.012–1.17 μM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.