Experimental annotation of the human genome using microarray technology.

The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.

The Leishmania genome project: new insights into gene organization and function.

The sequencing of Leishmania major Friedlin chromosome 1 (Chr1), Chr3, and Chr4 has been completed. and several other chromosomes are well underway. The complete genome sequence should be available by 2003. Over 1,000 full-length new genes have been identified, with the majority (approximately 75%) having unknown function. Many of these may be Leishmania (or kinetoplastid) specific. Most interestingly, the genes are organized into large (> 100-500 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a "divergent" manner, i.e., the mRNAs for the two sets of genes are both transcribed towards the telomeres. Nuclear run-on analysis suggests that transcription is initiated in both directions within the "divergent" region. Chr3 and Chr4 contain two "convergent" clusters, with a single "divergent" gene at one telomere of Chr3. Sequence analysis of several genes from the LD1 region of Chr35 indicates a high degree of sequence conservation between L. major and L. donovani/L. infantum within protein-coding open reading frames (ORFs), with a lower degree of conservation within the non-coding regions. Immunization of mice with recombinant antigen from two of these genes, BTI (formerly ORFG) and ORFF, results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

Genomic organization and gene function in Leishmania.

Sequencing of the Leishmania major Friedlin genome is well underway with chromosome 1 (Chr1) and Chr3 having been completely sequenced, and Chr4 virtually complete. Sequencing of several other chromosomes is in progress and the complete genome sequence may be available as soon as 2003. A large proportion ( approximately 70%) of the newly identified genes remains unclassified, with many of these being potentially Leishmania- (or kinetoplastid-) specific. Most interestingly, the genes are organized into large (>100-300 kb) polycistronic clusters of adjacent genes on the same DNA strand. Chr1 contains two such clusters organized in a 'divergent' manner, i. e. the mRNAs for the two sets of genes are both transcribed towards the telomeres. Chr3 contains two 'convergent' clusters, with a single 'divergent' gene at one telomere, with the two large clusters separated by a tRNA gene. We have characterized several genes from the LD1 (Leishmania DNA 1) region of Chr35. BT1 (formerly ORFG) encodes a biopterin transporter and ORFF encodes a nuclear protein of unknown function. Immunization of mice with recombinant antigens from these genes results in significant reduction in parasite burden following Leishmania challenge. Recombinant ORFF antigen shows promise as a serodiagnostic. We have also developed a tetracycline-regulated promoter system, which allows us to modulate gene expression in Leishmania.

The unusual gene organization of Leishmania major chromosome 1 may reflect novel transcription processes.

The complete chromosomal sequence for chromosome 1 from Leishmania major Friedlin predicts that this chromosome has 79 protein-coding genes. Surprisingly, the first 29 of these genes are encoded in tandem on one strand of DNA, and the remaining 50 genes are encoded in tandem on the other. No RNA polymerase promoters, centromeric sequences or origins of DNA replication have been identified in the DNA sequence. Statistical analyses of the nucleotide content reveal striking, non-random, sequence-biases that are correlated with genome organization. Analysis of coding regions suggests that novel transcription processes in Leishmania may be responsible for the nucleotide bias, which in turn affects gene organization in the chromosome. These results also suggest that the region between the two units of in-tandem genes is a candidate for an origin of DNA replication.

The role of tyrosine-9 and the C-terminal helix in the catalytic mechanism of Alpha-class glutathione S-transferases.

Glutathione S-transferases (GSTs) play a key role in the metabolism of drugs and xenobiotics. To investigate the catalytic mechanism, substrate binding and catalysis by the wild-type and two mutants of GST A1-1 have been studied. Substitution of the 'essential' Tyr(9) by phenylalanine leads to a marked decrease in the k(cat) for 1-chloro-2,4-dinitrobenzene (CDNB), but has no affect on k(cat) for ethacrynic acid. Similarly, removal of the C-terminal helix by truncation of the enzyme at residue 209 leads to a decrease in k(cat) for CDNB, but an increase in k(cat) for ethacrynic acid. The binding of a GSH analogue increases the affinity of the wild-type enzyme for CDNB, and increases the rate of the enzyme-catalysed conjugation of this substrate with the small thiols 2-mercaptoethanol and dithiothreitol. This suggests that GSH binding produces a conformational change which is transmitted to the binding site for the hydrophobic substrate, where it alters both the affinity for the substrate and the catalytic-centre activity ('turnover number') for conjugation, perhaps by increasing the proportion of the substrate bound productively. Neither of these two effects of GSH analogues are seen in the C-terminally truncated enzyme, indicating a role for the C-terminal helix in the GSH-induced conformational change.

Determinants of specificity for aflatoxin B1-8,9-epoxide in alpha-class glutathione S-transferases.

We have used homology modelling, based on the crystal structure of the human glutathione S-transferase (GST) A1-1, to obtain the three-dimensional structures of rat GSTA3 and rat GSTA5 subunits bound to S-aflatoxinyl-glutathione. The resulting models highlight two residues, at positions 208 and 108, that could be important for determining, either directly or indirectly, substrate specificity for aflatoxin-exo-8,9-epoxide among the Alpha-class GSTs. Residues at these positions were mutated in human GSTA1-1 (Met-208, Leu-108), rat GSTA3-3 (Glu-208, His-108) and rat GSTA5-5 (Asp-208, Tyr-108): in the active rat GSTA5-5 to those in the inactive GSTA1-1; and in the inactive human GSTA1-1 and rat GSTA3-3 to those in the active rat GSTA5-5. These studies show clearly that, in all three GSTs, an aspartate residue at position 208 is a prerequisite for high activity in aflatoxin-exo-8,9-epoxide conjugation, although this alone is not sufficient; other residues in the vicinity, particularly residues 103-112, are important, perhaps for the optimal orientation of the aflatoxin-exo-8,9-epoxide in the active site for catalysis to occur.

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution.

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.