Immune response of mice to Thy-1.1 antigen: Studies on congenic lines

The primary response to Thy-1.1 antigen was measured by a plaque assay that detected cells producing antibodies lytic for AKR thymocytes (PFC). TheH-2 congenic mice (B10.K and B10.BR) carryingH-2 complexes of high responders (CBA and C57BR) on the low-responder background (B10) were found to produce significantly fewer PFC than the corresponding donor of theH-2 complex. On the other hand, C3H.B10 mice carrying theH-2 complex of a low responder on the high-responder background produced significantly more PFC than the donor of theH-2 complex. These findings were interpreted as evidence that alleles at previously described loci believed to be components of theI region of theH-2 complex and controlling immune response to Thy-1.1 are influenced by alleles at another locus. Studies of segregating populations of theH-2 congenic lines supplied evidence that this locus, tentatively calledIr-5, is in chromosome 17 (linkage group IX).     

Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 s for the B800-850 complex, 14±2 s for the B850 complex incorporated with spheroidene, and 19±2 s for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 s. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49–55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.     

Restriction fragment length polymorphism and chromosome mapping of a mouse homeo box gene,Hox-2.1

Restriction endonuclease fragment length polymorphisms (RFLPs) were found using the cDNA probe Hox-2.1 for the homeo box-2.1 gene in the mouse. Polymorphism was detected in restriction patterns generated by fragments fromHindIII digestion. The great majority of laboratory strains of mice carries theHox-2.1 ^a allele. Only two laboratory strains carry theHox-2.1 ^b allele. Among strains of wild origin, the European subspecies (Mus m. domesticus, M. m. brevirostris, andM. m. musculus) and some Asian subspecies (M. m. castaneus) carry theHox-2.1 ^a allele. The subspecies from Far Eastern countries (M. m. molossinus, Chinese mice of wild origin, andM. m. yamashinai) carry theHox-2.1 ^ballele. Using the RFLP, theHox-2.1 gene was mapped on chromosome 11. Three-point cross test data showed that the recombination frequency is 29.6% between theHba and theHox-2.1 genes and 23.5% between theHox-2.1 and theEs-3 genes. The gene order ofHba-Hox-2.1-Es-3 has been confirmed.     

Characterization of five subgroups of the sieve element occlusion gene family in Glycine max reveals genes encoding non-forisome P-proteins,forisomes and forisome tails

P-proteins are structural phloem proteins discussed to be involved in the rapid sealing of injured sieve elements. P-proteins are found in all dicotyledonous and some monocotyledonous plants, but additional crystalloid P-proteins, known as forisomes, have evolved solely in the Fabaceae. Both types are encoded by members of the sieve element occlusion (SEO) gene family, which comprises seven phylogenetic subgroups. The Fabaceae-specific subgroup 1 contains genes encoding forisome subunits in e.g. Medicago truncatula, Vicia faba, Dipteryx panamensis and Canavalia gladiata whereas basal subgroup 5 encodes P-proteins in Nicotiana tabacum (tobacco) and Arabidopsis thaliana. The function of remaining subgroups is still unknown. We chose Glycine max (soybean) as a model to investigate SEO proteins representing different subgroups in one species. We isolated native P-proteins to determine the SEO protein composition and analyzed the expression pattern, localization and structure of the G. max SEO proteins representing five of the subgroups. We found that subgroup 1 GmSEO genes encode forisome subunits, a member of subgroup 5 encodes a non-forisome P-protein and subgroup 2 GmSEO genes encode the components of forisome tails, which are present in a restricted selection of Fabaceaen species. We therefore present the first molecular characterization of a Fabaceae non-forisome P-protein and the first evidence that forisome tails are encoded by a phylogenetically-distinct branch of the SEO gene family.     

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assembly of peripheral light-harvesting complexes in purple sulfur bacteria <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> ATCC 17899

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodopin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphenylamine (DPA) concentrations not exceeding 53 μM, while at illumination intensity decreased to 500 lx they could grow at 71 μM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoene synthetase (resulting in a decrease in the total carotenoid content) and of phytoene desaturase, which results in formation of neurosporene hydroxy derivatives and ζ-carotene. In the cells grown at 500 lx, small amounts of spheroidene and OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was ~15% of the control (without inhibition) for the B800-850 and ~20% of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with carotenoids due to disintegration of some carotenoidless complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.     

B lymphocyte alloantigens controlled by theI region of the major histocompatibility complex in mice

An antiserum was produced by reciprocal immunization of congenic resistent inbred strains of mice which differed only with respect to theI andS regions of theH- 2 complex. This antiserum permitted the serological detection of lymphocyte alloantigens, designated Ia (=I region associated antigens). Ia determinants are only present on mature B lymphocytes. They could not be found on thymocytes, splenic or lymph node T cells, or on the majority of bone marrow cells. Absorption studies demonstrated existence of several Ia specificities which are associated with differentI region types. Thus, theI region of theH- 2 complex appears to control not only T-cellexpressed antigen specific immune response genes, but also B-cell-expressed Ia determinants. The relevance of the Ia alloantigen system for cellular interaction in immune reactions is discussed.     

Analysis ofH-2 mutants: Evidence for multiple CML target specificities controlled by theH-2K b gene

C57BL/6 (H-2 ^b ) mice and two mutants derived from this strain, B6.C-H-2 ^ba (Hz1) andE6-H-2 ^bd (M505), were studied in a number of functional tests, in vitro and in vivo, that assay for differences at theH-2 complex. All three strains give rise to reciprocal mixed lymphocyte reactivity (MLR) and cell-mediated lympholysis (CML) in vitro as well as graft-host reactivity (GVHR) and skin graft rejection in vivo. Analysis for cross-reactivity between these strains in CML revealed that the gained antigens in each mutant do not cross-react, and that Hz1 has lost an antigen shared by C57BL/6 and M505 strains. In addition, spleen cells from B10.A(4R) mice, which differ from theH-2 ^b haplotype only at theK end of theH-2 complex, recognize a common antigen shared by all three strains tested. Provided that the mutations occurred in theH-2K ^b gene, these data indicate that a) there are at least three antigenic specificities coded for by theH-2K ^b gene(s) that serve as targets for receptors on thymus-derived (T) cells in CML; b) since C57BL/6 strain mice and the mutants are serologically indistinguishable on a qualitative basis, the antigens recognized by the receptors on T cells and by humoral H-2 antibody are nonidentical; and c) mutation in theH-2K ^b locus itself can give rise to allogeneic recognition phenomena such as MLR and GVHR.     

Expression inE. coli of the cloned cDNA for the major antigen of foot and mouth disease virus Asia 1 63/72

Double stranded cDNA for the foot and mouth disease virus was prepared, restricted withBamH 1 or ligated to linkers withBamH 1 sticky ends and cloned inBamH 1 site in the expression vector, pU R222. The cDNA was also cloned at thePst 1 site in the same vector by the dC/dG tailing method. They were transferred intoE. coli to give colourless colonies in the presence of the dye, X-gal. Many of them showed positive signal on hybridization with³²P-labelled viral RNA. The middleBamH1 fragment of the cDNA is known to carry the gene for the major antigen and some non-structural proteins. The clones carrying the recombinant DNA produced proteins which cross-reacted with the antibodies generated against the structural proteins of the virus in an enzyme linked immunosorbent assay, indicating that the cDNA of the major antigen is expressed in the cloned cell.     

An activating amino acid substitution in the c-abl oncogene protein fails to produce a local conformational change

Thebcr-abl chimeric gene of Philadelphia chromosome positive chronic myelogenous leukemias is only weakly transforming. This transformation activity is greatly enhanced by a Lys-for-Glu substitution at position 832 in the c-abl gene, as occurs in the highly transforming v-abl genes. It has been suggested that this mutation results in a significant structural change in the encoded protein product. Using conformational energy analysis, we have determined the allowed low-energy conformations for residues 828–836 of this protein with Lys and Glu at position 832. In both cases, the overwhelmingly preferred conformation for this region is a bend-helix motif. The helix terminates at residue 836, and there are no discernible differences in conformation between the Lys- and Glu-containing sequences. These results suggest that the activating amino acid substitution at position 832 in the c-abl protein product does not produce its effect via a local conformational change.     

Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

High salinity induced expression profiling of differentially expressed genes in shrimp (Penaeus monodon)

Four suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed salinity stress responsive genes of black tiger shrimp, Penaeus monodon exposed to high (55 ppt) salinity conditions. One each of the forward and reverse SSH cDNA libraries were developed from the gill and gut tissues of shrimp and clones having inserts larger than 300 bp were unidirectionally sequenced. Based on the sequence homology search, the identified genes were categorized for their putative functions related to a wide range of biological roles, such as nucleic acid regulation and replication, immune response, energy and metabolism, signal transduction, cellular process, structural and membrane proteins, stress and osmoregulation. Gene expression levels in response to high salinity conditions at 2 weeks post salinity stress for some of the differentially expressed genes (Na⁺/K⁺-ATPase α-subunit, glutathione peroxidase, intracellular fatty acid binding protein, elongation factor 2, 14-3-3 like protein, penaeidin, translationally controlled tumor protein, transglutaminase and serine proteinase inhibitor B3) identified from SSH cDNA libraries were analysed by real-time RT-PCR. The highest gene expression levels was observed for Na⁺/K⁺-ATPase α-subunit in gill tissues (15.23-folds) and antennal glands (12.01-folds) and intracellular fatty acid binding protein in gut tissues (14.05-folds) respectively. The differential and significant levels of gene expression indicate the functional role of these genes in shrimp salinity stress adaptive mechanisms.