Immunologic memory responses induced in BALB/c mice by cross-linked outer membrane extracts of four Salmonella serotypes.

Outer membrane proteins (OMP), extracted from Salmonella enteritidis, S anatum, S typhimurium, and S infantis, were cross-linked to form a large immunogen (4-OMP-lipopolysaccharide [LPS]). Vaccinations with 4-OMP-LPS dissolved in phosphate-buffered saline solution and 4-OMP-LPS emulsified with muramyl dipeptide were capable of eliciting specific and sustained primary IgM and IgG responses in BALB/c mice, as well as inducing immunologic memory for 130 days. In addition to 4-OMP-LPS-specific responses, substantial IgM and IgG responses specific for each live homologous organism were detected over the 130-day trial. In comparison with vaccination with 4-OMP-LPS dissolved in phosphate-buffered saline solution, responses specific for the antigen or the homologous Salmonella were not markedly increased in mice vaccinated with 4-OMP-LPS emulsified with muramyl dipeptide. Seemingly, cross-linked OMP, without the inclusion of muramyl dipeptide, may have potential as vaccine components and may induce immunologic memory.

Reduced Accumulation of ABA during Water Stress in a Molybdenum Cofactor Mutant of Barley.

A barley (Hordeum vulgare L.) mutant (Az34) has been identified with low basal levels of abscisic acid (ABA) and with reduced capacity for producing ABA in response to water stress. The mutation is in a gene controlling the molybdenum cofactor resulting in a pleiotropic deficiency in at least three molybdoenzymes, nitrate reductase, xanthine dehydrogenase, and aldehyde oxidase. The mutant was found to lack aldehyde oxidase activity with several substrates including: (a) ABA aldehyde, a putative precursor of ABA; (b) an acetylenic analog of ABA aldehyde; and (c) heptaldehyde. Elevating the growth temperature from 18 to 26 degrees C caused mutant leaves to wilt and brown. Desiccation of mutant leaves was prevented by applying ABA. These results indicate that ABA biosynthesis at some developmental stages is dependent upon a molybdoenzyme which may be an aldehyde oxidase.

Isolation, mapping, and characterization of two barley multiovary mutants.

Mutations in homeotic genes disturb the spatial and temporal patterns of development, often leading to the appearance of tissues in abnormal locations. Many homeotic genes, involved in flower development, code for proteins with a highly conserved domain called the MADS box, which acts as a sequence-specific DNA binding protein. Two floral development mutants were isolated from a fast neutron irradiated M2 barley population. The phenotypes are multiovary, that is, stamens replaced with carpels, designated mo7a, and stamens replaced with carpels and lodicules converted to leaflike structures, designated mo6b. These phenotypes resemble the Arabidopsis mutants APETALA3 (AP3) and PISTILATA (PI). The mo6b and mo7a mutants were mapped to the centromeric region of chromosome 1 (7H) and to the telomeric region of chromosome 3 (3H), respectively.

Association of the NAD(P)H-bispecific nitrate reductase structural gene with the Nar7 locus in barley.

The NADH-specific and NAD(P)H-bispecific nitrate reductase genes from barley have been cloned and sequenced. To determine if the Nar7 locus encodes the NAD(P)H-bispecific nitrate reductase structural gene, a cross was made between a wild-type cultivar, Morex (Nar7 Nar7), and Az70 (nar7w nar7w), a mutant from the cultivar Steptoe that is deficient in NAD(P)H-bispecific nitrate reductase activity. A probe specific to the NAD(P)H-bispecific nitrate reductase structural gene detected restriction fragment length polymorphism between the parents. This probe was used to classify selected F2 progeny for restriction fragment length genotype. All the NAD(P)H nitrate reductase deficient F2 progeny (24/101) possessed the Az70 restriction fragment genotype. The absence of recombination between the NAD(P)H-bispecific nitrate reductase deficient genotype and the NAD(P)H-bispecific nitrate reductase restriction fragment length genotype indicates that the two traits are closely associated in inheritance and that Nar7 is probably the NAD(P)H-bispecific nitrate reductase structural gene.

Immunoglobulin M and immunoglobulin G responses in BALB/c mice to conjugated outer membrane extracts of four Salmonella serotypes.

Outer membranes (OMs) of Salmonella enteritidis, S. anatum, S. typhimurium, and S. infantis were extracted and cross-linked with glutaraldehyde to form a large macromolecular antigen. The antigen consisted of OM proteins and lipopolysaccharide and was designated 4-OMP-LPS. Polyacrylamide gel electrophoresis of extracted OMs from each serotype revealed differences in protein profiles. S. enteritidis and S. infantis possessed a greater variety of proteins than did S. anatum and S. typhimurium. Immunizations with 4-OMP-LPS in phosphate-buffered saline (4-OMP-LPS-C) and 4-OMP-LPS emulsified with muramyl dipeptide in the oil phase of a hexadecane-water emulsion (4-OMP-LPS-MDP) revealed that BALB/c mice were capable of eliciting specific primary and secondary immunoglobulin M (IgM) and IgG responses. Both antigen preparations were capable of eliciting IgM and IgG specific for the cell surfaces of each live Salmonella serotype. Also, 4-OMP-LPS-MDP and 4-OMP-LPS-C were capable of evoking a substantial anamnestic response. Adsorption studies revealed that the combined serotypes had the antigenic capacity to adsorb up to 94% of the antibodies, but 4-OMP-LPS-MDP antibodies were more effectively adsorbed than were 4-OMP-LPS-C antibodies. Adsorption of pooled antiserum with heterologous bacteria yielded a variety of adsorption profiles.

Marker assisted genetic analysis of non-brittle rachis trait in barley.

Brittle rachis is a head shattering mechanism of barley. Two tightly linked complementary genes, btr1 and btr2, were believed to control the non-brittle rachis trait. Position of non-brittle rachis loci btr1btr2 on the short arm of Chromosome 3 was investigated using RFLP markers. Two approaches were employed. First, a Hordeum vulgare subsp. spontaneum fragment that confers brittleness in a cv. Bowman near isogenic line was detected. This fragment is 18-33 cM in length and contains MWG798B, ABG057, MWG014, BCD706 and KFP216 markers of the short arm of Chromosome 3. In the second approach, position of btr1 locus in a H. vulgare subsp. spontaneum (Wadi Qilt 23-38)xH. vulgare subsp. vulgare (cv. Harrington) cross was detected using a selective genotyping approach in BC2F1 generation. F-tests and analysis of genotypic compositions of BC2F1 lines showed that btr1 locus, and supposedly the tightly linked btr2 locus, is in 4.3 cM KFP216-RisP114 interval of short arm of Chromosome 3. Results also yielded clues for the presence of at least two additional loci that affect the non-brittle rachis trait. Allelism tests using genotypes with known non-brittle rachis gene compositions provided additional evidence for presence of such loci.

Rice-barley synteny and its application to saturation mapping of the barley Rpg1 region.

In order to facilitate the map-based cloning of the barley stem rust resistance gene Rpg1, we have demonstrated a high degree of synteny at a micro level between the telomeric region of barley chromosome 1P and rice chromosome 6. We have also developed and applied a simple and efficient method for selecting useful probes from large insert genomic YAC and cosmid clones. The gene order within the most terminal 6.5 cM of barley chromosome 1P was compared with the most terminal 2.7 cM of rice chromosome 6. Nine rice probes, previously mapped in rice or isolated from YAC or cosmid clones from this region, were mapped in barley. All, except one, were in synteny with the rice gene order. The exception, probe Y617R, was duplicated in barley. One copy was located on a different chromosome and the other in a non-syntenic position on barley chromosome 1P. The barley probes from this region could not be mapped to rice, but two of them were inferred to be in a syntenic location based on their position on a rice YAC. This work demonstrates the utility of applying the results of genetic and physical mapping of the small genome cereal rice to map-based cloning of interesting genes from large genome relatives.