A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

Interisland and interarchipelago transfer of stone tools in prehistoric Polynesia.

Tracing interisland and interarchipelago movements of people and artifacts in prehistoric Polynesia has posed a challenge to archaeologists due to the lack of pottery and obsidian, two materials most readily used in studies of prehistoric trade or exchange. Here we report the application of nondestructive energy-dispersive x-ray fluorescence (EDXRF) analysis to the sourcing of Polynesian artifacts made from basalt, one of the most ubiquitous materials in Polynesian archaeological sites. We have compared excavated and surface-collected basalt adzes and adze flakes from two sites in Samoa (site AS-13-1) and the Cook Islands (site MAN-44), with source basalts from known prehistoric quarries in these archipelagoes. In both cases, we are able to demonstrate the importing of basalt adzes from Tutuila Island, a distance of 100 km to Ofu Island, and of 1600 km to Mangaia Island. These findings are of considerable significance for Polynesian prehistory, as they demonstrate the movement of objects not only between islands in the same group (where communities were culturally and linguistically related) but also between distant island groups. Further applications of EDXRF analysis should greatly aid archaeologists in their efforts to reconstruct ancient trade and exchange networks, not only in Polynesia but also in other regions where basalt was a major material for artifact production.

A pre-Columbian Y chromosome-specific transition and its implications for human evolutionary history.

A polymorphic C-->T transition located on the human Y chromosome was found by the systematic comparative sequencing of Y-specific sequence-tagged sites by denaturing high-performance liquid chromatography. The results of genotyping representative global indigenous populations indicate that the locus is polymorphic exclusively within the Western Hemisphere. The pre-Columbian T allele occurs at > 90% frequency within the native South and Central American populations examined, while its occurrence in North America is approximately 50%. Concomitant genotyping at the polymorphic tetranucleotide microsatellite DYS19 locus revealed that the C-->T mutation displayed significant linkage disequilibrium with the 186-bp allele. The data suggest a single origin of linguistically diverse native Americans with subsequent haplotype differentiation within radiating indigenous populations as well as post-Columbian European and African gene flow. The mutation may have originated either in North America at a very early time during the expansion or before it, in the ancestral population(s) from which all Americans may have originated. The analysis of linkage of the DYS199 and the DYS19 tetranucleotide loci suggests that the C-->T mutation may have occurred around 30,000 years ago. We estimate the nucleotide diversity over 4.2 kb of the nonrecombining portion of the Y chromosome to be 0.00014. compared to autosomes, the majority of variation is due to the smaller effective population size of the Y chromosome rather than selective sweeps. There begins to emerge a pattern of pronounced geographical localization of Y-specific nucleotide substitution polymorphisms.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Synthetic zeolites and other microporous oxide molecular sieves

Use of synthetic zeolites and other microporous oxides since 1950 has improved insulated windows, automobile air-conditioning, refrigerators, air brakes on trucks, laundry detergents, etc. Their large internal pore volumes, molecular-size pores, regularity of crystal structures, and the diverse framework chemical compositions allow “tailoring” of structure and properties. Thus, highly active and selective catalysts as well as adsorbents and ion exchangers with high capacities and selectivities were developed. In the petroleum refining and petrochemical industries, zeolites have made possible cheaper and lead-free gasoline, higher performance and lower-cost synthetic fibers and plastics, and many improvements in process efficiency and quality and in performance. Zeolites also help protect the environment by improving energy efficiency, reducing automobile exhaust and other emissions, cleaning up hazardous wastes (including the Three Mile Island nuclear power plant and other radioactive wastes), and, as specially tailored desiccants, facilitating the substitution of new refrigerants for the ozone-depleting chlorofluorocarbons banned by the Montreal Protocol.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: Population genetics, human serologic response, and gene transcription

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase–PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae.

Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.

Carbonic anhydrase is an ancient enzyme widespread in prokaryotes

Carbonic anhydrases catalyze the reversible hydration of CO2 and are ubiquitous in highly evolved eukaryotes. The recent identification of a third class of carbonic anhydrase (γ class) in a methanoarchaeon and our present finding that the β class also extends into thermophilic species from the Archaea domain led us to initiate a systematic search for these enzymes in metabolically and phylogenetically diverse prokaryotes. Here we show that carbonic anhydrase is widespread in the Archaea and Bacteria domains, and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. The presence of the β and γ classes in metabolically diverse species spanning the Archaea and Bacteria domains demonstrates that carbonic anhydrases have a far more extensive and fundamental role in prokaryotic biology than previously recognized.

Smad proteins function as co-modulators for MEF2 transcriptional regulatory proteins

An emerging theme in transforming growth factor-β (TGF-β) signalling is the association of the Smad proteins with diverse groups of transcriptional regulatory proteins. Several Smad cofactors have been identified to date but the diversity of TGF-β effects on gene transcription suggests that interactions with other co-regulators must occur. In these studies we addressed the possible interaction of Smad proteins with the myocyte enhancer-binding factor 2 (MEF2) transcriptional regulators. Our studies indicate that Smad2 and 4 (Smad2/4) complexes cooperate with MEF2 regulatory proteins in a GAL4-based one-hybrid reporter gene assay. We have also observed in vivo interactions between Smad2 and MEF2A using co-immunoprecipitation assays. This interaction is confirmed by glutathione S-transferase pull-down analysis. Immunofluorescence studies in C2C12 myotubes show that Smad2 and MEF2A co-localise in the nucleus of multinuclear myotubes during differentiation. Interestingly, phospho-acceptor site mutations of MEF2 that render it unresponsive to p38 MAP kinase signalling abrogate the cooperativity with the Smads suggesting that p38 MAP Kinase-catalysed phosphorylation of MEF2 is a prerequisite for the Smad–MEF2 interaction. Thus, the association between Smad2 and MEF2A may subserve a physical link between TGF-β signalling and a diverse array of genes controlled by the MEF2 cis element.

Ectopic expression of the minimal whiE polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes

The single recombinant expressing the Streptomyces coelicolor minimal whiE (spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal whiE PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase whiE-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.

Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants

In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.

SET1, A Yeast Member of the Trithorax Family, Functions in Transcriptional Silencing and Diverse Cellular Processes

The trithorax gene family contains members implicated in the control of transcription, development, chromosome structure, and human leukemia. A feature shared by some family members, and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Here we present analysis of SET1, a yeast member of the trithorax gene family that was identified by sequence inspection to encode a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40–50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologues. To understand SET1 function(s), we created a null mutant. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure.

Diverse karyotypic abnormalities of the c-myc locus associated with c-myc dysregulation and tumor progression in multiple myeloma

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

Evolutionary relationships among diverse bacteriophages and prophages: All the world’s a phage

We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage φC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, φflu, in the Haemophilus influenzae genome, and two small prophage-like elements, φRv1 and φRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.