Evaluation of “sequence-tagged-site” PCR products as molecular markers in wheat

The polymerase chain reaction (PCR) is an attractive technique for many genome mapping and characterization projects. One PCR approach which has been evaluated involves the use of randomly amplified polymorphic DNA (RAPD). An alternative to RAPDs is the sequence-tagged-site (STS) approach, whereby PCR primers are designed from mapped low-copy-number sequences. In this study, we sequenced and designed primers from 22 wheat RFLP clones in addition to testing 15 primer sets that had been previously used to amplify DNA sequences in the barley genome. Our results indicated that most of the primers amplified sequences that mapped to the expected chromosomes in wheat. Additionally, 9 of 16 primer sets tested revealed polymorphisms among 20 hexaploid wheat genotypes when PCR products were digested with restriction enzymes. These results suggest that the STS-based PCR analysis will be useful for generation of informative molecular markers in hexaploid wheat.Contribution no. J-2833 of the Montana Agric Exp Stn     

High levels of diversity in Fusarium oxysporum from non-cultivated ecosystems in Australia

The Fusarium oxysporum species complex (FOSC) is a ubiquitous ascomycetous group that includes both pathogenic and non-pathogenic strains, the former being responsible for disease in over 100 cultivated plant species. Previous phylogenetic studies have uncovered at least four major clades within the FOSC, with Clade 1 hypothesised as being ancestral. However, the origin of these clades and pathogenic strains is poorly understood. Due to an emphasis on agricultural isolates in previous studies, the underlying diversity of this species complex in non-cultivated soils is largely unknown. To address this imbalance an extensive survey of isolates associated with native vegetation geographically isolated from cultivation throughout the Australian continent was conducted. A multi-gene phylogenetic analysis of the translation elongation factor (EF-1α) and the mitochondrial small subunit (mtSSU) rDNA loci did not recover any novel clades. However, the Australian isolates had high levels of intra-Clade diversity based on EF-1α sequence type (ST) comparison with a global dataset. The ST diversity was not equally distributed across the four clades, with the majority of novel STs recovered from Clade 1. Implications on the origin of the FOSC are discussed.     

Comparative proteomic analysis of virulent Korean Mycobacterium tuberculosis K-strain with other mycobacteria strain following infection of U-937 macrophage

In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDITOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.     

Enhancement of macrophage IL-1 production by Leishmania major infection in vitro and its inhibition by IFN-gamma

Peritoneal cells from highly susceptible BALB/c mice were infected with Leishmania major and cultured for various times in vitro. The culture supernatants contained significant levels of IL-1 which were consistently higher than those in the cell cultures stimulated with an optimal concentration of LPS. This finding extends to a macrophage cell line, P388D1, and peritoneal exudate cells stimulated with starch in vivo. However, the level of IL-1 produced was significantly reduced when the cells were preincubated with a lymphokine preparation (supernatant of Con A-stimulated rat spleen cells). The level of IL-1 produced seems to be directly correlated with the degree of parasitization of the macrophages. A similar and dose-dependent reduction in IL-1 production by infected macrophages could also be obtained when the cells were preincubated with IFN-gamma. This finding is in direct contrast to that of visceral leishmaniasis in which peritoneal macrophages from BALB/c mice infected with Leishmania donovani not only fail to produce IL-1 but also lose the capacity to produce IL-1. This apparent discrepancy is discussed in terms of a possible difference in the induction of cell-mediated immunity between the two leishmanial diseases.     

QTL analysis of cotton fiber length in advanced backcross populations derived from a cross between <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and <Emphasis Type="Italic">G. mustelinum</Emphasis>

Key message

QTLs for fiber length mapped in three generations of advanced backcross populations derived from crossing Gossypium hirsutum and Gossypium mustelinum showed opportunities to improve elite cottons by introgression from wild relatives.

Abstract

The molecular basis of cotton fiber length in crosses between Gossypium hirsutum and Gossypium mustelinum was dissected using 21 BC₃F₂ and 12 corresponding BC₃F_2:3 and BC₃F_2:4 families. Sixty-five quantitative trait loci (QTLs) were detected by one-way analysis of variance. The QTL numbers detected for upper-half mean length (UHM), fiber uniformity index (UI), and short fiber content (SFC) were 19, 20, and 26 respectively. Twenty-three of the 65 QTLs could be detected at least twice near adjacent markers in the same family or near the same markers across different families/generations, and 32 QTLs were detected in both one-way variance analyses and mixed model-based composite interval mapping. G. mustelinum alleles increased UHM and UI and decreased SFC for five, one, and one QTLs, respectively. In addition to the main-effect QTLs, 17 epistatic QTLs were detected which helped to elucidate the genetic basis of cotton fiber length. Significant among-family genotypic effects were detected at 18, 16, and 16 loci for UHM, UI, and SFC, respectively. Six, two, and two loci showed genotype?×?family interaction for UHM, UI and SFC, respectively, illustrating complexities that might be faced in introgression of exotic germplasm into cultivated cotton. Co-location of many QTLs for UHM, UI, and SFC accounted for correlations among these traits, and selection of these QTLs may improve the three traits simultaneously. The simple sequence repeat (SSR) markers associated with G. mustelinum QTLs will assist breeders in transferring and maintaining valuable traits from this exotic source during cultivar development.

     

Synthesis and biological studies of 5-aminolevulinic acid-containing dendrimers for photodynamic therapy.

Using a convergent growth approach, a series of novel 5-aminolevulinic acid (ALA)-containing dendrimers have been synthesized. In these molecules, ALA residues are attached to the periphery by ester linkages, with amide bonds connecting the dendrons. Three first-generation dendrimers, bearing either 6 or 9 ALA residues, were synthesized by attachment of a tris(Boc-protected ALA)-containing wedge (1) to a di- or tripodent aromatic, or tripodent aliphatic core. Two second generation 18-ALA-containing dendrimers were also synthesized using a 3,3'-iminodipropionic acid spacer unit between wedge 1 and the aromatic core. These compounds differed only in the distance between the core and the linker unit. The Boc-protected dendrimers were deprotected using trifluoroacetic acid and isolated as their TFA salts. The potential of these ALA ester dendrimers as macromolecular prodrugs for photodynamic therapy has been demonstrated in the tumorigenic keratinocyte PAM 212 cell line.     

Visual Impairment,Hearing Loss and Cognitive Function in an Older Population: Longitudinal Findings from the Blue Mountains Eye Study

The presence of visual impairment (VI) and hearing loss (HL) with may be a marker for subsequent cognitive decline over time in older people. A prospective, longitudinal population-based study of the 3654 participants of the Blue Mountains Eye Study were assessed for the associations between VI and HL and a decline in mini-mental state examination (MMSE) scores over a duration of 10 years from the 5-year (baseline of this report) to the 15-year follow-up visits. MMSE was assessed at the 5-, 10- and 15-year follow-up visits. A decline ≥3 scores from 5-year to 10- or 15-year visits indicated possible cognitive decline. VI was defined as best-corrected visual acuity <6/12 in the worse-eye, HL was defined as pure-tone average >40 decibels in the worse-ear and dual sensory impairment (DSI) was defined by the co-presence of VI and HL, detected at 5-year follow-up (baseline of this report). Participants with no VI and HL over the same 5- or 10-year corresponding period were controls. Associations of VI, HL and DSI with possible cognitive decline were assessed using logistic regression models adjusting for age and sex after excluding subjects with a stroke history. The presence of VI, HL or DSI was not associated with possible cognitive decline over 5 years (odds ratio (OR) 0.84, 95% confidence-intervals (CI) 0.40–1.79, OR 1.02, 95% CI 0.61–1.70 and 1.41, 95% CI 0.54–3.72, respectively) or 10 years (OR 1.09, 95% CI 0.52–2.30, OR 1.09, 95% CI 0.65–1.82 and 1.15, 95% CI 0.28–4.73, respectively). There were no changes to these findings after adjustment for other potential confounders. Age was significantly associated with possible cognitive decline (OR 1.07, 95% CI 1.04–1.10 for both periods). Neither visual impairment, hearing loss nor dual sensory impairment was independently associated with subsequent decline in cognition.     

Advances in the molecular biology of plant seed storage proteins

Plant seed storage proteins were among the first proteins to be isolated (20); however, only recently, as a result of using molecular biology techniques, have the amino acid sequences of many of these proteins been determined. With the accumulation of amino acid sequence data for many vicilin-type storage proteins much has been learned concerning the location of conserved amino acid regions and other regions which can tolerate amino acid sequence variation. Combining this knowledge with recent advances in plant gene transfer technologies will allow molecular biologists to correct (by using amino acid replacement mutations) the sulfur amino acid deficiency inherent to bean seed storage proteins. The development of more nutritious soybean and common bean seeds will be of benefit to programs involving human and animal nutrition.     

Effect of thyrotropin on the phosphorylation of thyroid chromosomal proteins.

Thyrotropin (TSH) stimulated the phosphorylation of histone H1 in calf thyroid slices but had no effect on other classes of histones. Phosphorylation of total phenol-soluble nonhistone chromosomal proteins was not affected by incubation with TSH. However, when these phenol-soluble nonhistone chromosomal proteins were analysed by two-dimensional gels involving isoelectrofocusing and dodecyl sulfate-polyacrylamide gel electrophoresis, TSH was shown to stimulate the phosphorylation of two specific groups of phosphoproteins with molecular weights between 35,000 and 45,000 and isoelectric points at pH values of 5.4-6.0. This increase in phosphorylation with TSH stimulation was confirmed by quantitative analysis of one-dimensional isoelectrofocusing gels.     

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.