Detection of an Ss-like protein in the sera of inbred and wild rats

A normal serum protein that crossreacts with rabbit anti-mouse Ss serum was isolated by alternating gel nitration and ion exchange chromatography from the inbred Long-Evans (LGE) rat strain. Rabbit antisera prepared against this protein detected it in the sera of all inbred and individual wild rats tested. The close physical and immunochemical similarity between this protein and the mouse C4 component of complement (Ss protein) indicates that this protein may represent the rat homolog of the mouse C4. Quantitative differences in the level of the Ss-like rat protein, comparable to those seen in Ss low mice, were not observed in 25 inbred strains or 22 individual wild rats. These quantitative results were supported by functional assays for total hemolytic complement and individual C2, C3, and C4 complement components. Sixteen inbred strains were examined and all had normal levels of activity for each of the assays.     

Sequence analysis of the complete mitochondrial DNA in 10 commonly used inbred rat strains

Rat remains a major biomedical model system for common, complex diseases. The rat continues to gain importance as a model system with the completion of its full genomic sequence. Although the genomic sequence has generated much interest, only three complete sequences of the rat mitochondria exist. Therefore, to increase the knowledge of the rat genome, the entire mitochondrial genomes (16,307–16,315 bp) from 10 inbred rat strains (that are standard laboratory models around the world) and 2 wild rat strains were sequenced. We observed a total of 195 polymorphisms, 32 of which created an amino acid change (nonsynonymous substitutions) in 12 of the 13 protein coding genes within the mitochondrial genome. There were 11 single nucleotide polymorphisms within the tRNA genes, six in the 12S rRNA, and 12 in the 16S rRNA including 3 insertions/deletions. We found 14 single nucleotide polymorphisms and 2 insertion/deletion polymorphisms in the D-loop. The inbred rat strains cluster phylogenetically into three distinct groups. The wild rat from Tokyo grouped closely with five inbred strains in the phylogeny, whereas the wild rat from Milwaukee was not closely related to any inbred strain. These data will enable investigators to rapidly assess the potential impact of the mitochondria in these rats on the physiology and the pathophysiology of phenotypes studied in these strains. Moreover, these data provide information that may be useful as new animal models, which result in novel combinations of nuclear and mitochondrial genomes, are developed. genome; mitochondria     

Immunologic enhancement of experimental metastasis in the rat

Summary Using a series of immunologically cross-reactive metastatic tumor variants, we demonstrate that serum from animals bearing pulmonary tumor colonies possesses enhancing properties in the experimental metastasis (lung colony) assay. Enhancement is produced by chronic serum administration and promotes the growth of tumor cells arrested in the lungs which would not otherwise proliferate to form grossly detectable lung nodules. Tumor-bearer serum from animals with lung colonies derived from the most highly metastatic variant examined is shown to possess enhancing properties in both BD-IX(H-1^d) and BD-IV(H-1^d) rat strains, while tumor-bearer serum from animals with lung colonies derived from the less metastatic parent tumor cell line possesses enhancing properties in the BD-IX rat strain only. Removal of immunoglobulin from enhancing serum by affinity column chromatography simultaneously removes the enhancing factor(s), and enhancing activity correlates with the presence of increased levels of Clq-binding immune complexes in the serum. Serum levels of immune complexes are shown to be more elevated in serum from animals bearing lung colonies derived from the most highly metastatic variant. The enhancing moieties are shown to bind to concanavalin A, but not to staphylococcal protein A, and the active fraction elutes from concanavalin A-Sepharose with -methyl-mannoside. Consideration of immunoprecipitation studies on whole and fractionated enhancing sera, along with studies on affinity purified isotype fractions reveals that the activity resides with antibodies of IgG_2b subclass. Abbreviations used: NK, natural killer cell; CIC, circulating immune complex; RhC, rheumatoid-Clq protein complex; Ig, immunoglobulin     

Polymorphism of the major histocompatibility locus in the wild Norway rat

Specific alloantisera against the eight Ag-B groups found in inbred strains of rats were capable of reacting with all wild Norway rats (Rattus norvegicus) tested. Absorption studies, antisera production, and progeny testing involving wild rats showed that the antigenic specificities detected in the wild rat population were similar, if not identical, to the Ag-B antigens present in inbred strains. Xenoantisera prepared in rabbits against rat erythrocyte antigens (Ag-C1 and/or C2) reacted with erythrocytes from each wild rat tested. Progeny testing involving these erythrocyte antigens was identical to that observed in inbred strains. The restricted genetic polymorphism of theAg-B alleles in the wild rat population suggests that the functional and evolutionary significance of the major histocompatibility complex in the rat may not depend upon a high degree of genetic variability.     

Use of the MHC for mate choice in wild house mice (Mus domesticus)

The mechanisms maintaining natural diversity at the major histocompatibility complex (MHC) are not well understood. To increase knowledge of one potential mechanism, I examined the use of MHC genes for mate choice by wild house mice in a controlled laboratory setting. Three rearing groups of wild test mice were produced: non‐fostered control mice, mice fostered into families of an inbred laboratory mouse strain, and mice fostered into families of a second, MHC‐congenic mouse strain. Mature test mice were given a choice of two opposite‐sex stimulus mice from the two MHC‐congenic strains used for fostering, and were scored for several measures of preference. The results were non‐significant in general, but females of two rearing groups spent significantly more time with mice of one MHC‐type, and in most rearing groups, mice tended to spend more time with this same MHC‐type. Other results showed that male test mice ejaculated indiscriminantly and that female wild mice mated to ejaculation more often in longer length trials, but showed no significant preferences. In this study, fostering seemed to have little or no effect on MHC‐based mate preferences of wild house mice, and wild mice did not appear to be using the MHC to avoid inbreeding. However, some wild female mice used the MHC to choose potential mates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ontogenesis and independent genetic control of the rat adenylate kinase isozymes (EC 2.7.4.3.)

The level of activity of cytoplasmic isozyme II of adenylate kinase (EC 2.7.4.3) appears to be genetically controlled in the rat (Rattus norvegicus). Adult animals of the Okamoto inbred strain exhibit a ninefold higher erythrocytic activity than the rats of the dilute agouti inbred strain. This difference seems to be due to two codominant autosomal alleles at a same locus. The study of the ontogeny of that enzyme in muscle in the two strains shows that the wellknown postnatal activity increase of that isozyme is reduced in the low activity (dilute agouti) strain. However, the mitochondrial isozyme III activity is not similarly regulated as no difference between the two strains has been observed.     

Linkage disequilibrium between two genetic loci of the major histocompatibility complex in rats

The major histocompatibility complex (MHC) in natural populations of rats is composed of genetic phenotypes that are similar, if not identical, to those seen in inbred laboratory strains. Examination of individual wild rats from a single location in the city of Pittsburgh, Pennsylvania, resulted in the identification of seven different RT1.A histocompatibility serotypes and three RT1.B mixed lymphocytes responses. In this population of animals there is a significant association (p < 0.005) between four RT1.A and RT1.B phenotypic pairs: RT1.A⁸B¹, RTl.A^kBⁿ, RTl.A^dB^a and RT1.A¹B^a. The observed values for linkage disequilibrium (0.211,0.076,0.070 and 0.085, respectively) are very high and are close to the maximum expected, given the individual allelic frequencies. Although the animals included in this study were obtained from one location, agreement with Hardy-Weinberg equilibrium is demonstrated for other loci in the same population. The demonstration of equilibrium suggests that significant inbreeding is not affecting this population of rats. Not enough is known about the allelic frequencies in surrounding rat populations to determine how important the effect of migration is on these disequilibrium values. The large linkage disequilibria may indicate that, in the rat, environmental selective forces are operating to ensure the nonrandom association of separate components of the MHC.     

Histocompatible chicken inbred lines: homogeneities in the major histocompatibility complex antigens of the GSP, GSN/1, PNP/DO and BM-C inbred lines assessed by hemagglutination, mixed lymphocyte reaction and skin transplantation.

Chicken inbred lines of the GSP, GSN/1, PNP/DO and BM-C have been established by selection of a specific allele at the B blood group locus (MHC B-G region) and other polymorphic loci through pedigree mating. To extend the potential of these inbred lines as experimental animals in Aves, we assessed the antigenic homogeneities of the MHC antigens by three immunological methods. Antigenic variations of red blood cells (RBCs) were surveyed in the inbred lines and a random-bred line (NG) derived from the Nagoya breed by using ten kinds of intact antisera produced in the inbred line of chickens against RBCs of a red junglefowl and hybrids. In the hemagglutination test, no individual variations were found within the inbred line at all, while all the ten antisera detected highly heterogeneous reactions in individuals of the NG. The reciprocal one-way mixed lymphocyte reactions gave constantly higher stimulation responses (P<0.01) between individual pairs from the inbred lines having different B alleles compared to pairs within the inbred line, while lower stimulation was observed between pairs of the GSP and GSN/1 inbred lines both having the B(21) allele. In reciprocal skin transplantation, the transplanted skingrafts within the inbred line and between individuals from the GSP and GSN/1 inbred lines survived more than 100 days, while all the skingrafts showed signs of rejection within 7 days among the inbred lines having different B alleles. The results obtained by the three practical methods coincidentally indicated that the individuals in the respective four inbred lines were histocompatible, and further, that the GSP and GSN/1 individuals were histocompatible.     

Derivation of a Germline Competent Transgenic Fischer 344 Embryonic Stem Cell Line

Embryonic stem (ES) cell-based gene manipulation is an effective method for the generation of mutant animal models in mice and rats. Availability of germline-competent ES cell lines from inbred rat strains would allow for creation of new genetically modified models in the desired genetic background. Fischer344 (F344) males carrying an enhanced green fluorescence protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. After establishment of ES cell lines, rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing was conducted in selected ES cell lines. One male ES cell line, F344-Tg.EC4011, was further evaluated for germline competence by injection into Dark Agouti (DA) X Sprague Dawley (SD) blastocysts. Resulting chimeric animals were bred with wild-type SD mates and germline transmissibility of the ES cell line was confirmed by identification of pups carrying the ES cell line-derived EGFP transgene. This is the first report of a germline competent F344 ES cell line. The availability of a new germline competent ES cell line with a stable fluorescence reporter from an inbred transgenic rat strain provides an important new resource for genetic manipulations to create new rat models.     

HLA-DR2-associated Dw subtypes correlate with RFLP clusters: most DR2 IDDM patients belong to one of these clusters

The incidence of arthritis and the antibody response to mouse and to rat type 11 collagen after immunization with native rat type II collagen was studied in different mouse strains, including wild mouse-derived strains belonging to the H-2^p/H-2^q family. High serum levels of antibodies to mouse and rat type II collagen were seen only in H-2^q mice, whereas mice belonging to the p, w3, w5, and w17 haplotypes displayed low type II collagen-specific antibody responses. Mice from three different H-2^q-carrying strains (DBA/1, NFR/N, and B10.G) with different non-major histocompatibility complex genes were all susceptible to collagen arthritis, but they displayed a varying incidence of arthritis and varying clinical features. No arthritis was seen in non-H-2^q mice, except in the B10.CAS2 strain where a few mice developed arthritis despite very low serum levels of type II collagen-specific antibodies. We conclude that small differences in the A chain of class II transplantation antigens are of importance for the development of arthritis and for the stimulation of a high response after immunization with type 11 collagen.Abbreviations used in this paper ELISA enzyme-linked immunosorbent assay - PBS phosphate-buffered saline - Ig immunoglobulin - MHC major histocompatibility complex     

Association of MHC class I and class II gene polymorphisms with vaccine or challenge response to Salmonella enteritidis in young chicks

Salmonella enteritidis (SE) is a worldwide source of salmonellosis in humans, caused mainly by consumption of contaminated poultry products. The major histocompatibility complex (MHC) class I and class II molecules, which function as antigen-presentation structures, are involved in both macrophage and dendritic cell immune response to Salmonella and may, therefore, play an important role in host resistance to infections. The chicken MHC class I and class II were investigated as candidate genes for immune response to SE. We characterized the complete MHC class I cDNA sequences for an outbred broiler line and four diverse highly inbred lines: two MHC-congenic Leghorn (G-B2 and G-B1), one Egyptian Fayoumi (M15.2), and one Spanish (Sp21.1), to define the allelic sequences within these lines, so that we might study the association between particular MHC polymorphisms and response to SE. The F1 offspring of outbred broiler sires crossed with three inbred lines (G-B1, G-B2, and Fayoumi) were evaluated as young chicks for either bacterial load in spleen and cecum after pathogenic SE inoculation or antibody level after SE vaccination. Alleles defined by a Lys(148)-->Met(148) polymorphism in the MHC class I alpha(2) domain were associated with spleen bacterial load after SE challenge. These results suggest that particular MHC haplotypes may contribute to control of responses to SE, and that particular polymorphisms may serve as markers for genetic resistance to SE in the chicken.     

Identification and profiling of novel microRNAs in the Brassica rapagenome based on small RNA deep sequencing

Background

Cowpea is a highly inbred crop. It is part of a crop-weed complex, whose origin and dynamics is unknown, which is distributed across the African continent. This study examined outcrossing rates and genetic structures in 35 wild cowpea (Vigna unguiculata ssp. unguiculata var. spontanea) populations from West Africa, using 21 isozyme loci, 9 of them showing polymorphism.

Results

Outcrossing rates ranged from 1% to 9.5% (mean 3.4%), which classifies the wild cowpea breeding system as primarily selfing, though rare outcrossing events were detected in each population studied. Furthermore, the analyses of both the genetic structure of populations and the relationships between the wild and domesticated groups suggest possibilities of gene flow that are corroborated by field observations.

Conclusions

As expected in a predominantly inbred breeding system, wild cowpea shows high levels of genetic differentiation and low levels of genetic diversity within populations. Gene flow from domesticated to wild cowpea does occur, although the lack of strong genetic swamping and modified seed morphology in the wild populations suggest that these introgressions should be rare.     

An increase in O-GlcNAcylation of Sp1 down-regulates the gene expression of pi class glutathione S-transferase in diabetic mice

Oxidative stress is a key factor contributing to the development of diabetes complications. Glutathione S-transferases (GSTs) protect against products of oxidative stress by conjugating glutathione to electrophilic substrates, producing compounds that are generally less reactive and more soluble. The expression and activity of GSTs during diabetes have been extensively studied, but little is known about regulation mechanisms of Pi-class GST (GSTP). The aim of the present study was to evaluate how GSTP is regulated in a Streptozotocin (STZ)-induced murine diabetes model. GST activity and GSTP expression were determined in adult male mice diabetized with STZ. Specificity protein 1 (Sp1) expression and O-glycosylation, as well as the role of AP-1 members Jun and Fos in the regulation of GSTP expression, were also assessed. The results showed that GST total activity and GSTP mRNA and protein levels were decreased in the diabetic liver, and returned to normal values after insulin administration. The insulin-mimetic drug vanadate was also able to restore GST activity, but failed to recover GSTP mRNA/protein levels. In diabetic animals, O-glycosylated Sp1 levels were increased, whereas, in insulin-treated animals, glycosylation values were similar to those of controls. After vanadate administration, Sp1 expression levels and glycosylation were lower than those of controls. Our results suggest that hyperglycemia could lead to the observed increase in Sp1 O-glycosylation, which would, in turn, lead to a decrease in the expression of Sp1-dependent GSTP in the liver of diabetic mice.     

Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo

We describe a novel insulin-degrading enzyme, SidC, that contributes to the proliferation of the human bacterial pathogen Vibrio vulnificus in a mouse model. SidC is phylogenetically distinct from other known insulin-degrading enzymes and is expressed and secreted specifically during host infection. Purified SidC causes a significant decrease in serum insulin levels and an increase in blood glucose levels in mice. A comparison of mice infected with wild type V. vulnificus or an isogenic sidC-deletion strain showed that wild type bacteria proliferated to higher levels. Additionally, hyperglycemia leads to increased proliferation of V. vulnificus in diabetic mice. Consistent with these observations, the sid operon was up-regulated in response to low glucose levels through binding of the cAMP-receptor protein (CRP) complex to a region upstream of the operon. We conclude that glucose levels are important for the survival of V. vulnificus in the host, and that this pathogen uses SidC to actively manipulate host endocrine signals, making the host environment more favorable for bacterial survival and growth.     

A novel strategy for genetic dissection of complex traits: the population of specific chromosome substitution strains from laboratory and wild mice

The mouse is an irreplaceable model for understanding the precise genetic mechanisms of mammalian physiological pathways. Thousands of quantitative trait loci (QTLs) have been mapped onto the mouse genome during the last two decades. However, only a few genes’ underlying complex traits have been successfully identified, and rapid fine mapping of QTL genes still remains a challenge for mouse geneticists. Currently, the Collaborative Cross (CC) has proceeded to the goal of establishing more than 1,000 recombinant inbred strains for the sub-centimorgan mapping resolution of QTL loci. In this article, a novel complementary strategy, designated as population of specific chromosome substitution strains or PSCSS, is proposed for rapid fine mapping of QTLs on the substituted chromosome. One specific chromosome (Chr 1) of recipient mouse strain C57BL/6 J has been substituted by homologous counterparts from five different inbred strains (C3H/He, FVB/N, AKR, NOD/LtJ, NZW/LacJ), an outbred line Kunmin mouse in China, and 23 wild mice captured in different localities. The primary genetic studies on the structure of these wild donor chromosomes (Chr 1) show that these donor chromosomes harbor extensive genetic polymorphisms, with a high density of SNP distribution, abundant variations of STR alleles, and a high level of historical recombination accumulation. These specific chromosome substitution strains eventually form a special population that has the identical genetic background of the recipient strain and differs only in the donor chromosomes. With simple association studies, known QTLs on the donor chromosome can be rapidly mapped in high resolution without requirement of further crosses. This approach, taking advantage of the extensive genetic polymorphisms of wild resources and chromosome substitution strategy, brings a new outlook for genetic dissection of complex traits.     

Genomic Determinants of Triglyceride and Cholesterol Distribution into Lipoprotein Fractions in the Rat

The plasma profile of major lipoprotein classes and its subdivision into particular fractions plays a crucial role in the pathogenesis of atherosclerosis and is a major predictor of coronary artery disease. Our aim was to identify genomic determinants of triglyceride and cholesterol distribution into lipoprotein fractions and lipoprotein particle sizes in the recombinant inbred rat set PXO, in which alleles of two rat models of the metabolic syndrome (SHR and PD inbred strains) segregate together with those from Brown Norway rat strain. Adult male rats of 15 PXO strains (n = 8–13/strain) and two progenitor strains SHR-Lx (n = 13) and BXH2/Cub (n = 18) were subjected to one-week of high-sucrose diet feeding. We performed association analyses of triglyceride (TG) and cholesterol (C) concentrations in 20 lipoprotein fractions and the size of major classes of lipoprotein particles utilizing 704 polymorphic microsatellite markers, the genome-wide significance was validated by 2,000 permutations per trait. Subsequent in silico focusing of the identified quantitative trait loci was completed using a map of over 20,000 single nucleotide polymorphisms. In most of the phenotypes we identified substantial gradient among the strains (e.g. VLDL-TG from 5.6 to 66.7 mg/dl). We have identified 14 loci (encompassing 1 to 65 genes) on rat chromosomes 3, 4, 7, 8, 11 and 12 showing suggestive or significant association to one or more of the studied traits. PXO strains carrying the SHR allele displayed significantly higher values of the linked traits except for LDL-TG and adiposity index. Cholesterol concentrations in large, medium and very small LDL particles were significantly associated to a haplotype block spanning part of a single gene, low density lipoprotein receptor-related protein 1B (Lrp1b). Using genome-wide association we have identified new genetic determinants of triglyceride and cholesterol distribution into lipoprotein fractions in the recombinant inbred panel of rat model strains.     

Glycerol-3-phosphate dehydrogenase expression and oxygen consumption in liver mitochondria of female and male rats with chronic alteration of thyroid status

In our chronic experiments (over several months), the activity and protein amount of glycerol-3-phosphate dehydrogenase (GPDH) in mitochondria isolated from the liver of adult male and female inbred Lewis strain euthyroid (EU), hyperthyroid (TH), and hypothyroid (HY) rats were analyzed by biochemical and Western blot methods. The TH status was induced by intraperitoneal injections of 3,3',5-triiodo- L-thyronine and the HY status with 0.05% solution of methimazole in drinking water. The TH status led to a significant increase and the HY status to a significant decrease of enzyme activity and protein amount in both male and female animals. These changes were, however, more pronounced in females. The EU and TH female rats also showed a significantly higher activity and the TH female rats showed also a significantly higher enzyme amount in comparison with males, while the HY rats showed low levels in both sexes. The glycerol-3-phosphate-dependent oxygen consumption of freshly isolated rat liver mitochondria from the TH animals was higher in comparison with the EU animals and it was activated by idebenone, a synthetic analogue of coenzyme Q, in both the EU and TH rats. Measurements of serum thyroid hormone levels and analysis of anatomical parameters (relative heart and thyroid gland weights) confirmed that our procedures inducing the TH and HY states are efficient and reliable and that determination of GPDH can serve as an additional criterion for the evaluation of the thyroid hormone status.     

Genetic analyses of alloreactions between recently wild and classical inbred strains of syrian hamsters: Evidence in favor of a major histocompatibility complex

Cytotoxic alloantisera were raised between recently wild and classical inbred strains of Syrian hamsters. Antisera produced by immunizing the classical inbred strains with tissue from the partially inbred, recently wild hamsters detect several specificities shared between the classical and recently wild strains. Reciprocal mixed lymphocyte reactions between the two different groups of hamsters suggest that the new source of hamsters possesses several unique MLR phenotypes which may represent new Hm-1 haplotypes. Moreover, several recently wild strains express MLR phenotypes quite similar if not identical to the Hm-1 ^a haplotype of the inbred strain, MHA. Genetic analyses of alloreactions between domestic inbred and recently wild strains suggest that a single locus or chromosomal region encodes the allodeterminants that induce strong MLR reactivity. Six unique MLR phenotypes have been defined which most likely represent haplotypes of the hamster MHC equivalent, Hm-1. Genetic linkage studies indicate that some alloantisera detect determinants encoded by loci closely linked to the MLR locus, and therefore define Hm-1 determinants. Moreover, other alloantisera recognize determinants encoded by a locus that is unlinked to Hm-1. These studies suggest that Syrian hamsters express a polymorphic MHC equivalent, Hm-1, which encodes determinants that induce both cell-mediated and humoral alloreactivity.