Optimizing purebred selection for crossbred performance using QTL with different degrees of dominance

A method was developed to optimize simultaneous selection for a quantitative trait with a known QTL within a male and a female line to maximize crossbred performance from a two-way cross. Strategies to maximize cumulative discounted response in crossbred performance over ten generations were derived by optimizing weights in an index of a QTL and phenotype. Strategies were compared to selection on purebred phenotype. Extra responses were limited for QTL with additive and partial dominance effects, but substantial for QTL with over-dominance, for which optimal QTL selection resulted in differential selection in male and female lines to increase the frequency of heterozygotes and polygenic responses. For over-dominant QTL, maximization of crossbred performance one generation at a time resulted in similar responses as optimization across all generations and simultaneous optimal selection in a male and female line resulted in greater response than optimal selection within a single line without crossbreeding. Results show that strategic use of information on over-dominant QTL can enhance crossbred performance without crossbred testing.     

Effect of zinc deficiency on the mRNA expression pattern in liver and jejunum of adult rats: monitoring gene expression using cDNA microarrays combined with real-time RT-PCR

In the study presented here, the effect of zinc deficiency on mRNA expression levels in liver and jejunum of adult rats was analyzed. Feed intake was restricted to 8 g/day. The semi-synthetic diet was fortified with pure phytate and contained either 2 μg Zn/g (Zn deficiency, n = 6) or 58 μg Zn/g (control, n = 7). After 29 days of Zn depletion feeding, entire jejunum and liver were retrieved and total RNA was extracted. Tissue specific expression pattern were screened and quantified by microarray analysis and verified individually via real-time RT-PCR. A relative quantification was performed with the newly developed Relative Expression Software Tool © on numerous candidate genes which showed a differential expression.

This study provides the first comparative view of gene expression regulation and fully quantitative expression analysis of 35 candidate genes in a non-growing Zn deficient adult rat model. The expression results indicate the existence of individual expression pattern in liver and jejunum and their tissue specific regulation under Zn deficiency. In addition, in jejunum a number of B-cell related genes could be demonstrated to be suppressed at Zn deficiency. In liver, metallothionein subtype 1 and 2 (MT-1 and MT-2) genes could be shown to be dramatically repressed and therefore represent putative markers for Zn deficiency. Expression results imply that some genes are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis.     

Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination

Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Intensive Linkage Mapping in a Wasp (Bracon Hebetor) and a Mosquito (Aedes Aegypti) with Single-Strand Conformation Polymorphism Analysis of Random Amplified Polymorphic DNA Markers

The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a diploid mosquito, Aedes aegypti. RAPD-SSCP analysis revealed segregation of codominant alleles at markers that appeared to segregate as dominant (band presence/band absence) markers or appeared invariant on agarose gels. Our SSCP protocol uses silver staining to detect DNA fractionated on large thin polyacrylamide gels and reveals more polymorphic markers than agarose gel electrophoresis. In B. hebetor, 79 markers were mapped with 12 RAPD primers in six weeks; in A. aegypti, 94 markers were mapped with 10 RAPD primers in five weeks. Forty-five percent of markers segregated as codominant loci in B. hebetor, while 11% segregated as codominant loci in A. aegypti. SSCP analysis of RAPD-PCR markers offers a rapid and inexpensive means of constructing intensive linkage maps of many species.     

Molecular biodiversity of cassava begomoviruses in Tanzania: evolution of cassava geminiviruses in Africa and evidence for East Africa being a center of diversity of cassava geminiviruses

Cassava is infected by numerous geminiviruses in Africa and India that cause devastating losses to poor farmers. We here describe the molecular diversity of seven representative cassava mosaic geminiviruses (CMGs) infecting cassava from multiple locations in Tanzania. We report for the first time the presence of two isolates in East Africa: (EACMCV-[TZ1] and EACMCV-[TZ7]) of the species East African cassava mosaic Cameroon virus, originally described in West Africa. The complete nucleotide sequence of EACMCV-[TZ1] DNA-A and DNA-B components shared a high overall sequence identity to EACMCV-[CM] components (92% and 84%). The EACMCV-[TZ1] and -[TZ7] genomic components have recombinations in the same genome regions reported in EACMCV-[CM], but they also have additional recombinations in both components. Evidence from sequence analysis suggests that the two strains have the same ancient origin and are not recent introductions. EACMCV-[TZ1] occurred widely in the southern part of the country. Four other CMG isolates were identified: two were close to the EACMV-Kenya strain (named EACMV-[KE/TZT] and EACMV-[KE/TZM] with 96% sequence identity); one isolate, TZ10, had 98% homology to EACMV-UG2Svr and was named EACMV-UG2 [TZ10]; and finally one isolate was 95% identical to EACMV-[TZ] and named EACMV-[TZ/YV]. One isolate of African cassava mosaic virus with 97% sequence identity with other isolates of ACMV was named ACMV-[TZ]. It represents the first ACMV isolate from Tanzania to be sequenced. The molecular variability of CMGs was also evaluated using partial B component nucleotide sequences of 13 EACMV isolates from Tanzania. Using the sequences of all CMGs currently available, we have shown the presence of a number of putative recombination fragments that are more prominent in all components of EACMV than in ACMV. This new knowledge about the molecular CMG diversity in East Africa, and in Tanzania in particular, has led us to hypothesize about the probable importance of this part of Africa as a source of diversity and evolutionary change both during the early stages of the relationship between CMGs and cassava and in more recent times. The existence of multiple CMG isolates with high DNA genome diversity in Tanzania and the molecular forces behind this diversity pose a threat to cassava production throughout the African continent.