Mapping quantitative trait loci for downy mildew resistance in pearl millet

Quantitative trait loci (QTLs) for resistance to pathogen populations of Scelerospora graminicola from India, Nigeria, Niger and Senegal were mapped using a resistant x susceptible pearl millet cross. An RFLP map constructed using F₂ plants was used to map QTLs for traits scored on F₄ families. QTL analysis was carried out using the interval mapping programme Mapmaker/QTL. Independent inheritance of resistance to pathogen populations from India, Senegal, and populations from Niger and Nigeria was shown. These results demonstrate the existence of differing virulences in the pathogen populations from within Africa and between Africa and India. QTLs of large effect, contributing towards a large porportion of the variation in resistance, were consistently detected in repeated screens. QTLs of smaller and more variable effect were also detected. There was no QTLs that were effective against all four pathogen populations, demonstrating that pathotype-specific resistance is a major mechanism of downy mildew resistance in this cross. For all but one of the QTLs, resistance was inherited from the resistant parent and the inheritance of resistance tended to be the result of dominance or over-dominance. The implications of this research for pearl millet breeding are discussed.     

Human Microtubule-Associated Protein-2c Localizes to Dendrites and Axons in Fetal Spinal Motor Neurons

Abstract: Microtubule-associated protein-2 (MAP-2) functions to maintain neuronal morphology by promoting the assembly of microtubules. MAP-2c is an alternately spliced form of MAP-2, containing the first 151 amino acids of high-molecular-weight (HMW) MAP-2 joined to the last 321 amino acids, eliminating 1,352 amino acids specific to HMW MAP-2. A polyclonal antibody generated to the splice site of human MAP-2c was used to determine its cellular localization. The MAP-2c antiserum was depleted of any HMW MAP-2 reactivity by absorption with HMW MAP-2 fusion protein. Western blot analysis of human fetal spinal cord homogenates demonstrated that the antibody is specific for human MAP-2c. MAP-2c immunoreactivity was found in the perinuclear cytoplasm and processes of anterior motor neurons and large processes of the posterior column in sections from 22–24-week human fetal spinal cord. Double-label confocal microscopy was performed using the MAP-2c polyclonal antibody and either a HMW MAP-2 or a neurofilament protein (highly phosphorylated 160- and 200-kDa protein) monoclonal antibody to identify these processes as dendrites or axons, respectively. HMW MAP-2 and MAP-2c colocalized in cell bodies and dendrites of anterior motor neurons, demonstrating for the first time the presence of native MAP-2c within dendrites. In addition, immunoelectron microscopy showed MAP-2c associated with microtubules in dendrites of motor neurons. MAP-2c and the neurofilament proteins were found in axons of the dorsal and ventral roots. The presence of MAP-2c within axons and dendrites suggests that MAP-2c contributes to neuronal plasticity during human fetal development.     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.     

Effects of UV-B on flavonoids, ferulic acid, growth and photosynthesis in barley primary leaves

Barley (Hordeum vulgare L.) was grown with UV-B (280–320 nm) at levels simulating 25 nr 5% ozone depletion on the date of the summer solstice al 40°N latitude, with UV-A (320–400 nm), or with no supplemental irradiation. In plant growth chambers providing 300 μmol m^?2 s^?1 photosynthetically active radiation (PAR). UV-B-grown leaves elongated more slowly than controls but reached the same final length 1 day later. Leal specific fresh weight (mass leaf area^?1) was significantly increased by UV-B after the 7th day of growth. IV-B did not significantly affect leaf area, fresh weight, dry weight, total chlorophylls, total carotenoids or photosynthetic quantum efficiency. CO₂ assimilation was decreased by UV-B only at internal CO₂ levels above 250 μl l^?1. By the 8th day of growth, UV-B increased flavonoid (saponarin and lutonarin) accumulation in both the lower epidermis and the mesophyll: about 40% of the saponarin and 20% of the lutonarin were in the lower epidermis under all experimental conditions. Glasshouse conditions proved too variable for reproducible determination of growth and photosynthesis but were reliable for determining developmental changes in flavonoid (saponarin and lutonarin) accumulation and provided up to 800 μmol m^?2 s^?1 PAR. In the glasshouse UV-B-grown leaves had more flavonoids than controls al all stages from 5 to 30 days after planting: ca 509 more saponarin and 100% more lutonarin. Levels of soluble (vacuolar) ferulic acid esters were similar under all conditions on day 5. and on day 20 or later, but were significantly higher in UV-B-grown plants on days 10 and 15. UV-B decreased insoluble (cell-wall-bound) ferulic acid esters on a whole leaf basis but significantly increased this fraction in the lower epidermis. UV-A had no significant effects on growth, photosynthesis or ferulic acid, but it slightly increased flavonoid accumulation. The results are discussed in terms of secondary phenolics as a tissue-specific, developmentally regulated adaptive response to UV-B.     

The Impact of Chlorophyll-Retention Mutations,d1d2 and cyt-G1, during Embryogeny in Soybean

The ultrastructural, physiological, and molecular changes in developing and mature seeds were monitored in a control line (Glycine max [L.] Merr., cv Clark) that exhibited seed degreening and two mutant lines (d1d2 and cyt-G1) that retained chlorophyll upon seed maturation. Ultrastructural studies showed that the control line had no internal membranes, whereas stacked thylakoid membranes were detected in the green seed from the mutant lines. Pigment analyses indicated that total chlorophyll was lowest in the mature seeds of the control line. Mature d1d2 and cyt-G1 seed had elevated Chl a and Chl b levels, respectively. In both control and mutant lines, Lhcb1, Lhcb2, and RbcS mRNAs were abundant in embryos prior to cotyledon filling, declined after the onset of storage protein accumulation, and were barely detectable or undetectable in all later stages of seed development. Therefore, the chlorophyll-retention phenotype must be a result of the alteration of a process that occurs after translation of photosynthesis-related mRNAs to stabilize apoprotein and pigment levels. Furthermore, different elements controlling either the synthesis or turnover of Chl a and Chl b must be impaired in the d1d2 and cyt-G1 lines. No reproducible differences in total leaf, embryonic, and chloroplast protein profiles and plastid DNAs could be correlated with the mutations that induced chlorophyll retention.     

A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

Analysis of randomly isolated cDNAs from developing endosperm of rice (Oryza sativa L.): evaluation of expressed sequence tags,and expression levels of mRNAs

Using a cDNA library prepared from poly(A)⁺ RNA from 10-day-old rice endosperm, partial nucleotide sequences of randomly isolated clones were analyzed. A total of 153 (30.6%) out of 500 cDNA clones showed high amino acid identity to previously identified genes. There was significant redundancy in cDNAs encoding prolamine and glutelin. About 21.0% of the cDNA clones were found to code for seed storage protein genes. Consequently, 37 independent genes were identified. Using cDNA clones encoding glutelin, prolamine, seed allergen, -1,4-glucan branching enzyme, glycine-rich RNA binding protein, metallothionein, non-specific lipid-transfer protein and ubiquitin conjugating enzyme the accumulation of mRNA during rice seed development was compared. Genes associated with seed storage protein and starch biosynthesis were expressed according to expected developmental stages. Glycinerich RNA binding protein genes as well as metallothionein-like protein genes were highly expressed in developing seeds, but low in leaves of whole plants.     

Cloning and expression of the Vitreoscilla hemoglobin gene in pseudomonads: effects on cell growth

The gene (vgb) encoding the hemoglobin (VtHb) of Vitreoscilla sp. was cloned into a broad-host-range vector and stably transformed into Pseudomonas putida, Pseudomonas aeruginosa, and Xanthomonas maltophilia. vgb was stably maintained and expressed in functional form in all three species. When growth of the P. aeruginosa and X. maltophilia transformants in Luria-Bertani medium was compared with that of each corresponding untransformed strain, the VtHb-producing strains reached slightly higher maximum viable cell numbers, had significantly increased viability after extebded times in culture, and, like E. coli that produces VtHb, had significantly lower respiration rates. The VtHb-producing strain of P. putida also reached a slightly higher maximum viable cell number than its corresponding untransformed strain, but was significantly less viable after extended times in culture and, unlike the case in E. coli, had a generally higher respiration rate than the untransformed strain. When growth was monitored by absorbance, the results were similar to those obtained with viable cell counts.     

云南普通马矮型马蛋白多态性及其品种分化关系

本文运用蛋白电泳技术对来自云南省文山州马关县和麻栗坡县的２１匹普通马和１４匹矮型马进行了分析。共分析遗传座位４４个,其中有１０个座位检测到多态性。根据分子钟假说和相应的公式,推算两者的分歧时间约为１８．５万年。     

Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase.

Mutants of Saccharomyces cerevisiae lacking a functional SOD1 gene encoding Cu/Zn superoxide dismutase (SOD) are sensitive to atmospheric levels of oxygen and are auxotrophic for lysine and methionine when grown in air. We have previously shown that these defects of SOD-deficient yeast cells can be overcome through mutations in either the BSD1 or BSD2 (bypass SOD defects) gene. In this study, the wild-type allele of BSD1 was cloned by functional complementation and was physically mapped to the left arm of chromosome VII. BSD1 is identical to PMR1, encoding a member of the P-type ATPase family that localizes to the Golgi apparatus. PMR1 is thought to function in calcium metabolism, and we provide evidence that PMR1 also participates in the homeostasis of manganese ions. Cells lacking a functional PMR1 gene accumulate elevated levels of intracellular manganese and are also extremely sensitive to manganese ion toxicity. We demonstrate that mutations in PMR1 bypass SOD deficiency through a mechanism that depends on extracellular manganese. Collectively, these findings indicate that oxidative damage in a eukaryotic cell can be prevented through alterations in manganese homeostasis.