Sexual reproduction and gene flow in the pine pathogen Dothistroma septosporum in British Columbia.

Dothistroma septosporum has caused a serious needle blight epidemic in the lodgepole pine forests in northwest British Columbia over the past several years. Although ascocarps had been observed in British Columbia, nothing was known about the contribution of sexual reproduction, gene flow and long-distance dispersal to the epidemic. Amplified fragment length polymorphism and mating-type markers in 19 sites were used to generate population and reproductive data. Overall, evidence suggests a mixed mode of reproduction. Haplotypic diversity was high, with 79 unique and 56 shared haplotypes (possible clones) identified from 192 fungal isolates. Overall, mating-type segregation did not differ significantly from 1:1; however, random mating was rejected in most populations in the index of association and parsimony tree-length permutation analyses using the full data set and, when using clone-corrected data sets, more of the smaller populations showed random mating. Two of the smaller populations consistently showed random mating for both tests using both clone-corrected and noncorrected data. High gene flow is suggested by no differentiation between 14 of the 19 sites, several of which came from young plantations where the pathogen was not likely present prior to the current outbreak. The remaining five sites showed some level of divergence, possibly due to historic separation and endemic pathogen populations. Results indicate a high evolutionary potential and long-distance dispersal in this pathogen, important to consider in future forest management.

Phylogeography and conservation genetics of Lake Qinghai scaleless carp Gymnocypris przewalskii.

The objective of this study was to examine the spatial genetic relationships of the Lake Qinghai scaleless carp Gymnocypris przewalskii within the Lake Qinghai system, determining whether genetic evidence supports the current taxonomy of Gymnocypris przewalskii przewalskii and Gymnocypris przewalskii ganzihonensis and whether Gymnocypris przewalskii przewalskii are returning to their natal rivers to spawn. Comparison of mitochondrial (control region) variation (42 haplotypes in 203 fish) of G. przewalskii with the postulated ancestral species found in the Yellow River, Gymnocypris eckloni (10 haplotypes in 23 fish), indicated no haplotype sharing, but incomplete lineage sorting. Consistent with the sub-species status, an AMOVA indicated that the Ganzi River population was significantly different from all other river populations (F(ST) = 0·1671, P < 0·001). No genetic structure was found among the other rivers in the Lake Qinghai catchment. An AMOVA of amplified fragment length polymorphism (AFLP) loci, however, revealed significant genetic differences between most spawning populations (F(ST) = 0·0721, P < 0·001). Both mitochondrial and AFLP data found significant differences among G. p. przewalskii, G. p. ganzihonensis and G. eckloni (F(ST) values of 0·1959 and 0·1431, respectively, P < 0·001). Consistent with the incomplete lineage sorting, Structure analysis of AFLP loci showed evidence of five clusters. One cluster is shared among all sample locations, one is unique to G. p. ganzihonensis and G. eckloni, and the others are mostly found in G. p. przewalskii. Genetic evidence therefore supports the current taxonomy, including the sub-species status of G. p. ganzihonensis, and is consistent with natal homing of most Lake Qinghai populations. These findings have significant implications for the conservation and management of this unique and threatened species. The evidence suggests that G. p. przewalskii should be treated as a single population for conservation purposes. Exchangeability of the populations, however, should not be used to promote homogenization of fish spawning in the different rivers. As some degree of genetic divergence was detected in this study, it is recommended that the spawning groups be treated as separate management units.

PAK signaling in oncogenesis.

The p21-activated kinase (PAK) family of serine/threonine kinases is important in physiological processes including motility, survival, mitosis, transcription and translation. PAKs are evolutionally conserved and widely expressed in a variety of tissues and are often overexpressed in multiple cancer types. Depending on structural and functional similarities, the six members of PAK family are divided into two groups with three members in each group. Group I PAKs are activated by extracellular signals through GTPase-dependent and GTPase-independent mechanisms. In contrast, group II PAKs are constitutively active. Over the years, accumulating data from tissue culture models and human tumors has increased our understanding about the biology of PAK family members. In this review, we have summarized the complex regulation of PAK and its downstream diverse myriads of effectors, which in turn are responsible for the biological effects of PAK family of kinases in cancer cells.

Isolation and characterization of 16 microsatellite loci in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae: Scolytinae).

We isolated 16 polymorphic microsatellite loci in the mountain pine beetle (Dendroctonus ponderosae Hopkins) and developed conditions for amplifying these markers in four multiplex reactions. Three to 14 alleles were detected per locus across two sampled populations. Observed and expected heterozygosities ranged from 0.000 to 0.902 and from 0.100 to 0.830, respectively. Three loci deviated from Hardy-Weinberg equilibrium in one sampled population. One of these loci may be sex linked. These markers will be useful in the study of population structure in this important pest species.

Genome organization of glutamine synthetase genes in rainbow trout (Oncorhynchus mykiss).

Unlike mammals, bony fish appear to possess multiple genes encoding glutamine synthetase (GS), the nitrogen metabolism enzyme responsible for the conversion of glutamate and ammonia into glutamine at the expense of ATP. This study reports on the development of genetic markers for each of the four isoforms identified thus far in rainbow trout (Oncorhynchus mykiss) and their genome localization by linkage mapping. We found that genes coding for GS01, GS02, GS03, and GS04 map to four different linkage groups in the trout genome, namely RT-24, RT-23, RT-08, and RT-13, respectively. Linkage groups RT-23 and RT-13 appear to represent distinct chromosomes sharing duplicated marker regions, which lends further support to the previous suggestion that GS02 and GS04 may be duplicate gene copies that evolved from a whole-genome duplication in the trout ancestor. In contrast, there is at present no further evidence that RT-24 and RT-08 share ancestrally homologous segments and additional genomic studies will be needed to clarify the evolutionary origin of genes coding for GS01 and GS03.

SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase.

Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K(i) = 0.19 microM). SP600125 is a reversible ATP-competitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-gamma, TNF-alpha, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharide-induced expression of tumor necrosis factor-alpha and inhibited anti-CD3-induced apoptosis of CD4(+) CD8(+) thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

Mechanistic effects of autophosphorylation on receptor tyrosine kinase catalysis: enzymatic characterization of Tie2 and phospho-Tie2.

Activation of receptor tyrosine kinases by autophosphorylation is one of the most common and critical transformations in signal transduction, yet its role in catalysis remains controversial. Autophosphorylation of the angiogenic receptor tyrosine kinase Tie2 was studied in terms of the autophosphorylation sites, sequence of phosphorylation at these sites, kinetic effects, and mechanistic consequences. Isoelectric focusing electrophoresis and mass spectrometric analysis of a Tie2 autophosphorylation time course showed that Tyr992 on the putative activation loop was phosphorylated first followed by Tyr1108 in the C-terminal tail (previously unidentified autophosphorylation site). Autophosphorylation of Tie2 to produce pTie2 resulted in a 100-fold increase in k(cat) and a 460-fold increase in k(cat)/K(m). Viscosity studies showed that the unphosphorylated Tie2 was partially limited by product diffusion ((k(cat))(eta) = 0.67 +/- 0.06), while product release was more rate-limiting ((k(cat))(eta) = 0.94 +/- 0.08) for autophosphorylated Tie2 (pTie2). Furthermore, autophosphorylation did not significantly affect the phosphoacceptor dissociation constants. There was a significant (k(cat))(H)/(k(cat))(D) solvent isotope effect (SIE) for unphosphorylated Tie2 (2.42 +/- 0.12) and modest SIE (1.28 +/- 0.04) for pTie2, which is consistent with the chemistry step being more rate-limiting for Tie2 as compared to pTie2. The pH-rate profiles of Tie2 and pTie2 revealed a >0.5 unit shift in the pK(a) values of catalytically relevant ionizable residues upon autophosphorylation. The shift in rate-limiting step will result in a different distribution of enzyme pools (e.g., E, E*S, E*P, etc.) which may modulate the susceptibility to inhibition. Tie2 and pTie2 were profiled with a panel of known ATP-competitive kinase inhibitors. Tie2 activation perturbs catalytic residue ionizations, shifts the rate-limiting step to almost exclusive diffusion-control, and transforms the kinase into a more perfect catalyst.

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping.

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.

Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases.

An effective procedure for the synthesis of 2-deoxy-2-fluoro-sugar nucleotides via Select fluor-mediated electrophilic fluorination of glycals with concurrent nucleophilic addition or chemo-enzymatic transformation has been developed, and the fluorinated sugar nucleotides have been used as probes for glycosyltransferases, including fucosyltransferase III, V, VI, and VII, and sialyl transferases. In general, these fluorinated sugar nucleotides act as competitive inhibitors versus sugar nucleotide substrates and form a tight complex with the glycosyltransferase.

Major histocompatibility complex class II A genes in cichlid fishes: identification, expression, linkage relationships, and haplotype variation.

Two cichlid species, the haplochromine Aulonocara hansbaenschi and the tilapiine Oreochromis niloticus, were used to study the major histocompatibility complex (Mhc) class II A variation within this group. Multiple class II A sequences were recovered from A. hansbaenschi and O. niloticus cDNA libraries and three sequence families, DAA, DBA, and DCA, were identified. Sets of O. niloticus haploid embryo families were used to determine the linkage relationships of these genes. Two independently assorting linkage groups were detected, DAA and DBA/DCA, neither of which is linked to the previously described Mhc class I gene cluster. Three DCA genes and up to four DBA genes were found to segregate in different haplotypes, whereas DAA occurred as a single locus. Four DBA haplotypes, DBA*H1-H4, were identified and shown to co-segregate with the previously described class II B haplotypes. Four DCA haplotypes, DCA*H1-H4, were found at a distance of 37 cM from the DBA/class II B cluster; in one DCA haplotype, DCA*H5, the genes were tightly linked to the DBA/class II B clusters. Transcripts of DAA and DBA genes were found in O. niloticus hepatopancreas and spleen; transcripts of DCA genes were detected in the A. hansbaenschi cDNA library, but not in O. niloticus. These findings provide a basis for using class II haplotypes as markers in the study of adaptive radiation in the cichlid species flocks of the East African Great Lakes.

Identification and linkage of the proteasome activator complex PA28 subunit genes in zebrafish.

PA28 is an activator of the latent 20S proteasome, a large multisubunit complex involved in intracellular proteolysis. Two forms of hexameric PA28 have been identified, PA28-(alphabeta)3 and PA28-(gamma)6, of which the former is of immunological importance. Both the PA28-alpha and PA28-beta subunits are inducible by interferon-gamma (IFN-gamma) and the PA28-(alphabeta)3 complex enhances the ability of the 20S proteasome to produce peptides suited for binding to major histocompatibility complex (Mhc) class I molecules. To identify the homologues of the PA28 subunits in zebrafish we screened a cDNA library and obtained full-length cDNA sequences of the genes PSME1, PSME2 and PSME3 coding for the PA28-alpha, PA28-beta and PA28-gamma subunits, respectively. Phylogenetic analysis indicates the existence of the ancestors of all three genes prior to the divergence of tetrapods and bony fishes. The IFN-gamma-inducible subunits, PA28-alpha and PA28-beta, evolve faster than the presumably older PA28-gamma subunit. Using zebrafish radiation hybrid panels, the genes PSME2 and PSME3 were mapped to linkage group 12 and shown to be separated by a distance of less than 2.4 cM. This observation suggests that an intrachromosomal duplication event created the precursor of the IFN-gamma-inducible genes from a PA28-gamma-like ancestor prior to their recruitment into the Mhc class I peptide presentation pathway.

Identification of seven genes in the major histocompatibility complex class I region of the zebrafish.

Physical linkage of genes whose products are involved in similar physiological pathways may have functional significance. The identification of conserved gene linkage in distantly related organisms can therefore strengthen the hypothesis of selection acting towards keeping genes on a chromosome. We used the cDNA selection technique and the polymerase chain reaction (PCR) with generic primers for the identification of new genes on the genomic clones bearing the major histocompatibility complex (Mhc) class I genes of the zebrafish (Danio rerio). We found six new genes (BING1, DAXX, TAPBP, KNSL2, TAP2B and KE6) whose orthologues are known to be linked to the Mhc class II region in humans and mice. In addition, a new zebrafish Mhc class I gene, termed Dare-UFA, was detected. By contrast, a search for the human leucocyte antigen (HLA)-linked BING3, KE3 and SACM2L genes revealed that these loci are not located on the class I clones of the zebrafish. The zebrafish class I region contains repetitive elements with similarity to the DANA, SATA and LINE repeats, as well as Tc1 transposable elements. Our findings indicate a high degree of linkage conservation between the zebrafish class I and the mammalian class II regions.

A contig map of the Mhc class I genomic region in the zebrafish reveals ancient synteny.

In contrast to the human and mouse Mhc, in which the clusters of class I and class II loci reside in close vicinity to one another, in the zebrafish, Danio rerio, they are found in different linkage groups. Chromosome walking using BAC (bacterial artificial chromosome) and PAC (P1 artificial chromosome) clones reveals the zebrafish class I region to occupy a segment of approximately 450 kb and to encompass at least 19 loci. These include three class I (Dare-UDA, -UEA, -UFA), five proteasome subunit beta (PSMB8, -9A, -9C, -11, -12), two TAPs (TAP2A, TAP2B), and one TAP binding protein (TAPBP). This arrangement contrasts with the arrangements found in human and mouse Mhc, in which the orthologues of the PSMB, TAP, and TAPBP loci reside within the class II region. In addition to this main zebrafish class I contig, a shorter contig of about 150 kb contains two additional class I (UBA, UCA) and at least five other loci. It probably represents a different haplotype of part of the class I region. The previously identified UAA gene shares an identical 5' part with UEA, but the two genes differ in their 3' parts. One of them is probably the result of an unequal crossing over. The described organization has implications for the persistence of syntenic relationships, coevolution of loci, and interpretation of the origin of the human/mouse Mhc organization.

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes.

In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked.

Analysis of a 26-kb region linked to the Mhc in zebrafish: genomic organization of the proteasome component beta/transporter associated with antigen processing-2 gene cluster and identification of five new proteasome beta subunit genes.

Sequencing of zebrafish (Danio rerio) bacterial artificial chromosome and P1 artificial chromosome genomic clone fragments and of cDNA clones has led to the identification of five new loci coding for beta subunits of proteasomes (PSMB). Together with the four genes identified previously, nine PSMB genes have now been defined in the zebrafish. Six of the nine genes reside in the zebrafish MHC (Mhc) class I region, four of them reside in a single cluster closely associated with TAP2 on a 26-kb long genomic fragment, and two reside at some distance from the fragment. In addition to homologues of the human genes PSMB5 through PSMB9, two new genes, PSMB11 and PSMB12, have been found for which there are no known corresponding genes in humans. The new genes reside in the PSMB cluster in the Mhc. Homology and promoter region analysis suggest that the Mhc-associated genes might be inducible by IFN-gamma. The zebrafish class I region contains representatives of three phylogenetically distinguishable groups of PSMB genes, X, Y, and Z. It is proposed that these genes were present in the ancestral PSMB region before Mhc class I genes became associated with it.

IkappaB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.

Activation of the transcription factor NF-kappaB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IkappaB. We recently purified a large multiprotein complex, the IkappaB kinase (IKK) signalsome, which contains two regulated IkappaB kinases, IKK1 and IKK2, that can each phosphorylate IkappaBalpha and IkappaBbeta. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFkappaB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IkappaB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-kappaB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-kappaB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-kappaB activation.

Isolation of mhc class II DMA and DMB cDNA sequences in a marsupial: the gray short-tailed opossum (Monodelphis domestica).

We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the alpha-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified.

Sequence variation at the major histocompatibility complex DRB loci in beluga (Delphinapterus leucas) and narwhal (Monodon monoceros).

The variation at loci with similarity to DRB class II major histocompatibility complex loci was assessed in 313 beluga collected from 13 sampling locations across North America, and 11 narwhal collected in the Canadian high Arctic. Variation was assessed by amplification of exon 2, which codes for the peptide binding region, via the polymerase chain reaction, followed by either cloning and DNA sequencing or single-stranded conformation polymorphism analysis. Two DRB loci were identified in beluga: DRB1, a polymorphic locus, and, DRB2, a monomorphic locus. Eight alleles representing five distinct lineages (based on sequence similarity) were found at the beluga DRB1 locus. Although the relative number of alleles is low when compared with terrestrial mammals, the amino acid variation found among the lineages is moderate. At the DRB1 locus, the average number of nonsynonymous substitutions per site is greater than the average number of synonymous substitutions per site (0.0806 : 0.0207, respectively; P<0.01). Most of the 31 amino acid substitutions do not conserve the physiochemical properties of the residue, and 21 of these are located at positions implicated as forming pockets responsible for the selective binding of foreign peptide side chains. Only DRB1 variation was examined in 11 narwhal, revealing a low amount of variation. These data are consistent with an important role for the DRB1 locus in the cellular immune response of beluga. In addition, the ratio of nonsynonymous to synonymous substitutions is similar to that among primate alleles, arguing against a reduction in the balancing selection pressure in the marine environment. Two hypotheses may explain the modest amount of Mhc variation when compared with terrestrial mammals: small population sizes at speciation or a reduced neutral substitution rate in cetaceans.