Recent advances in large-scale transposon mutagenesis.

Transposons were identified as mobile genetic elements over fifty years ago and subsequently became powerful tools for molecular-genetic studies. Recently, transposon-mutagenesis strategies have been developed to identify essential and pathogenicity-related genes in pathogenic microorganisms. Also, a number of in vitro transposition systems have been used to facilitate genome sequence analysis. Finally, transposon mutagenesis of yeast and complex eukaryotes has provided valuable functional genomic information to complement genome-sequencing projects.

Wisconsin Card Sorting Test performance in patients with focal frontal and posterior brain damage: effects of lesion location and test structure on separable cognitive processes.

Forty-six patients with single focal lesions (35 frontal, 11 nonfrontal) were administered the Wisconsin Card Sorting Test (WCST) under three conditions of test administration. The three conditions varied in the amount of external support provided via specificity of instructions. The WCST, while a multifactorial test, is specifically sensitive to the effects of frontal lobe damage if deficits in language comprehension and visual-spatial search are controlled. There is also specificity of functioning within the frontal lobes: patients with inferior medial frontal lesions, unilateral or bilateral, were not impaired on the standard measures although they had increased loss of set when informed of the sorting categories. Verbal instructions may provide a probe to improve diagnosis and prognosis, assessment of the potential efficacy of treatment, and the time frame of plasticity of specific cognitive operations.

Structure-activity relationships of four 11-hydroxyestrones isomeric at the C-9 and C-11 positions.

The synthesis of 11 alpha-hydroxyestrone, 11 alpha-hydroxy-9 beta-estrone, and 11 beta-hydroxy-9 beta-estrone are presented. The reduction of 11-keto-9 beta-estrone 17-ethyleneketal by sodium in ethanol or sodium borohydride resulted in 11-hydroxy-9 beta-estrones. The 11-hydroxyl group configurations were opposite to expectations: sodium in boiling ethanol afforded the axial 11 beta-hydroxy-9 beta-estrone, while sodium borohydride in boiling tetrahydrofuran gave the equatorial 11 alpha-hydroxy-9 beta-estrone. In immature rat uterotropic bioassays using subcutaneous injections, 11 alpha-hydroxyestrone was 2 times as active as 11 alpha-hydroxy-9 beta-estrone, and 11 beta-hydroxyestrone was 10 times as active as 11 beta-hydroxy-9 beta-estrone.

The musculoskeletal manifestations of progeria. A literature review.

Although a superficial similarity exists between the musculoskeletal disorders associated with natural aging and those of progeria, an in-depth analysis reveals profound differences in the pathophysiology between the two processes. The protean manifestations of progeria can best be explained on the basis of the vascular changes found at autopsy. A disorder of the vascular endothelium may predispose progeric vessels to atherosclerotic changes. The unique musculoskeletal manifestations of progeria arise from the effects of premature atherosclerosis on the vascularized connective tissues.

Treatment of cystic fibrosis in adults.

More patients with cystic fibrosis are surviving into adulthood. Primary care physicians need a basic understanding of adult cystic fibrosis and the evaluation of patients with acute decompensation. Respiratory decompensation is usually the result of infective agents, including Pseudomonas aeruginosa, Stentrophomonas maltophilia and Burkholderia cepacia, and requires treatment with intravenous antibiotics. If symptoms worsen, the possibility of another complication, such as pneumothorax, hemoptysis, mycobacterial infection and allergic bronchopulmonary aspergillosis, must be considered. Family physicians can play an important role in the birth to adult care of patients with cystic fibrosis.

Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations.

An intrinsic feature of yeast artificial chromosomes (YACs) is that the cloned DNA is generally in the same size range (i.e., approximately 200-2000 kb) as the endogenous yeast chromosomes. As a result, the isolation of YAC DNA, which typically involves separation by pulsed-field gel electrophoresis, is frequently confounded by the presence of a comigrating or closely migrating endogenous yeast chromosome(s). We have developed a strategy that reliably allows the isolation of any YAC free of endogenous yeast chromosomes. Using recombination-mediated chromosome fragmentation, a set of Saccharomyces cerevisiae host strains was systematically constructed. Each strain contains defined alterations in its electrophoretic karyotype, which provide a large-size interval devoid of endogenous chromosomes (i.e., a karyotypic "window"). All of the constructed strains contain the kar1-delta 15 mutation, thereby allowing the efficient transfer of a YAC from its original host into an appropriately selected window strain using the kar1-transfer procedure. This approach provides a robust and efficient means to obtain relatively pure YAC DNA regardless of YAC size.

The Aspergillus nidulans sepA gene encodes an FH1/2 protein involved in cytokinesis and the maintenance of cellular polarity.

Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature-sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila. Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion mutants of sepA exhibit temperature-sensitive growth and severe delays in septation at the permissive temperature, indicating that expression of another gene may compensate for the loss of sepA. Conidiophores formed by sepA mutants exhibit abnormal branching of the stalk and vesicle. These results suggest that sepA interacts with the actin cytoskeleton to promote formation of the actin ring during cytokinesis and that sepA is also required for maintenance of cellular polarity during hyphal growth and asexual morphogenesis.

Regions of microsynteny in Magnaporthe grisea and Neurospora crassa.

A bacterial artificial chromosome (BAC) clone containing 110,467 bp of genomic DNA from Magnaporthe grisea was sequenced, annotated, and compared to the genomes of Neurospora crassa, Candida albicans, and Saccharomyces cerevisiae. Twenty-six open reading frames (ORFs), involved in multiple biochemical pathways, were identified in the BAC sequence. A region of 53 kb, containing 18 of the 26 ORFs, was found to be syntenic to a portion of the N. crassa genome. Subregions of complete colinearity as well as interrupted colinearity were present. No synteny was evident with either C. albicans or S. cerevisiae. The identification of syntenic regions containing highly conserved genes across two genera that have been evolutionarily separated for approximately 200 million years elicits many biological questions as to the function and identity of these genes.

The effects of focal anterior and posterior brain lesions on verbal fluency.

Seventy-four patients with focal brain lesions were compared to a neurologically normal control group on tasks of letter-based and category-based list generation. When patients were divided only by right frontal, left frontal, or nonfrontal lesion sites, the pattern of fluency impairments confirmed prior claims. When more precise lesion sites within the frontal lobes were compared between groups classified based on their fluency performance, much more specific brain-behavior relations were uncovered. Damage to the right dorsolateral cortical or connecting striatal regions, the right posterior area, or the medial inferior frontal lobe of either hemisphere did not significantly affect letter-based fluency performance. Superior medial frontal damage, right or left, resulted in moderate impairment. Patients with left dorsolateral and/or striatal lesions were most impaired. Left parietal damage led to performance relatively equivalent to the superior medial and left dorsolateral groups. The same lesion sites produced impairments in category based fluency, but so did lesions of right dorsolateral and inferior medial regions. Task analysis and correlations with other measures revealed that different cognitive processes related to different brain regions underlie performance on verbal fluency tests.

Gene discovery and gene function assignment in filamentous fungi.

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.