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PubMed2002

Molecular immunogenetics in susceptibility to bovine dermatophilosis: a candidate gene approach and a concrete field application.

To identify molecular genetic markers of resistance or susceptibility to dermatophilosis in cattle, we used a functional candidate gene approach to analyze the DNA polymorphisms of targeted genes encoding molecules implicated in known mechanisms of both nonspecific and specific immune responses existing in the pathogen/host interface mechanisms. The most significant results were obtained within the Major Histocompatibility Complex (MHC) where the BoLA-DRB3 and DQB genes encode molecules involved in the antigen presentation to T cell receptors. A unique BoLA class II haplotype, made up of one DRB3 exon 2 allele and one DQB allele, highly correlates with the susceptibility character (P < 0.001). This haplotype marker of susceptibility was also found and validated in other bovine populations. A eugenic marker-assisted selection was developed in the field by eliminating only the animals having this haplotype. The disease prevalence was thereby reduced from 0.76 to 0.02 over 5 years. A crossbreeding plan is in progress to study the genetic transmission of the genotypic and phenotypic characters of susceptibility to dermatophilosis. In conclusion, we discuss several hypotheses at the molecular and cellular levels to better define the exact role of the MHC molecules in disease control and to answer the question: How is MHC diversity selectively maintained by natural selection imposed by pathogens?

Actinomycetales Infections↗
PubMed1995

Growth phase-dependent induction of stationary-phase promoters of Escherichia coli in different gram-negative bacteria.

RSF1010-derived plasmids carrying a fusion of a promoterless lacZ gene with the sigma s-dependent growth phase-regulated promoters of Escherichia coli, bolAp1 and fic, were constructed. The plasmids were mobilized into the gram-negative bacterial species Acetobacter methanolicus, Xanthomonas campestris, Pseudomonas putida, and Rhizobium meliloti. The beta-galactosidase activities of bacterial cultures were determined during exponential and stationary growth phases. Transcriptional activation of the fic promoter in the different bacteria was growth phase dependent as in E. coli and was initiated generally during the transition to stationary phase. The induction of the bolA promoter was also growth phase dependent in the bacteria tested. While the expression in E. coli and R. meliloti was initiated during the transition from exponential to stationary phase, the induction in A. methanolicus, P. putida, and X. campestris started some hours after stationary growth phase was reached. In all the species tested, DNA fragments hybridizing with the rpoS gene of E. coli were detected. The results show that in different gram-negative bacteria, stationary-phase-specific sigma factors which are structurally and functionally homologous to sigma s and are able to recognize the promoter sequences of both bolA and fic exist.

Bacterial Proteins↗
PubMed1986

Use of bovine lymphocyte antigens in diagnosis of parentage.

Lymphocyte antigens controlled by alleles at the BoLA-A locus were used to indicate which of two bulls could be eliminated as the sire in 18 cases of disputed parentage. The use of bovine lymphocyte antigens (BoLA) significantly increased the exclusion probability over that of the standard red cell and electrophoretic techniques.

Animals↗
PubMed1999

Physical assignment of the bovine MHC class IIa and class IIb genes.

Screening of a bovine yeast artificial chromosome (YAC) library revealed two clones which contain most of the class II genes of the major histocompatibility complex (MHC) known to date. The YACs were mapped by fluorescence in situ hybridization (FISH) and characterized for the class II genes they contain. We found that the classic class II genes BoLA- DQA, -DQB, -DRA, and -DRB3 are located at BTA 23q21 and the non-classic class II genes DYA, DIB, LMP2, LMP7, TAP2, BoLA-DOB, -DMA, -DMB, and -DNA are located at BTA 23q12-->q13. These two different mapping locations confirm and extend previous findings of a gross physical distance between classic and non-classic MHC class II genes in cattle.

Animals↗
PubMed1995

Differences in bovine lymphocyte antigen associations between immune responsiveness and risk of disease following intramammary infection with Staphylococcus aureus.

This study evaluated the relationships between immune response, disease resistance, and bovine leukocyte antigens, BoLA, in Holstein cows following intramammary challenge with Staphylococcus aureus. This investigation was to determine whether immune responsiveness differed between these cows and whether differences were related to expression of class I BoLA antigens, which might explain the increased resistance or susceptibility to S. aureus mastitis. Antibody responses to S. aureus in milk and serum, total IgG1 in milk, and blastogenic responses with and without concanavalin A were evaluated. The CA42 allele, previously associated with increased risk of infection, was relatively uninformative for the immune response parameters examined. Other alleles, such as W3, were associated with higher milk antibody responses to S. aureus and higher milk IgG1 postchallenge. Alleles W7, W4, and W26 were associated with lower milk IgG1 and lower antibody titers in serum postchallenge. The association reported between allele CA42 and increased risk of S. aureus mastitis did not relate to lower antibody or blastogenic responses by these cows; however, indicators of innate resistance were not examined. In addition, the different associations detected between milk and serum emphasize the importance of considering mucosal immunity and unique immunological compartments when searching for relevant genetic markers of disease resistance.

Animals↗
PubMed1991

Bovine T cells recognize antigen in association with MHC class II haplotypes defined by one-dimensional isoelectric focusing.

A recently established, one-dimensional isoelectric focusing (IEF) method for distinguishing major histocompatibility complex (MHC) class II polymorphisms in an outbred species, cattle, has allowed us to analyse the involvement of the MHC in the recognition of antigen by bovine T cells. Bovine T-cell lines of Th cell phenotype (BoCD4+) specific for ovalbumin were generated from six individual high responder animals. These animals were bovine MHC (BoLA) class II typed using the IEF technique which detects bovine DR-like products. Four of the animals were shown to be heterozygous and two were homozygous for the IEF specificities. Six out of the 13 IEF specificities (EDF types) detected so far were represented by this group of animals. The cell lines were tested against a panel of IEF-typed antigen-presenting cells (APC) from unrelated donors. The lines only responded to antigen in proliferation assays when the APC shared at least one MHC class II EDF specificity with the BoCD4+ cell line. The responses did not correlate with BoLA class I specificities. However, lines from one of the animals were consistently generated to one of the two haplotypes only. This suggests that there are non-responder alleles to a multi-epitope antigen, present in the cattle population. The results demonstrate that IEF of bovine MHC class II products defines haplotypes of functional relevance, and may indeed be identifying the actual restriction elements involved in presentation of ovalbumin. These results have important implications for future vaccine design in an outbred species, particularly in terms of immune response gene effects and disease associations.

Animals↗
PubMed1984

Structure of thrombospondin.

The NH2-terminal amino acid sequences of thrombospondin and of a 30,000-Da heparin-binding peptide derived from thrombospondin by treatment with plasmin are identical. The heparin-binding peptide is homogeneous in size but slightly heterogeneous in charge with the predominant isoelectric points being 6.1 and 5.7. Electron microscopy of tungsten replicas of thrombospondin reveals a tripartite structure resembling a "bola" which is about 60 nm across when fully extended. Each part of the molecule terminates in a globular node or head which disappears upon limited plasmin digestion, suggesting that the heparin-binding peptide is located in the head region. In addition to the heparin-binding peptide, a 20,000-Da peptide also apparently associated with the head region is liberated during proteolysis. The electron micrographs indicate that the legs of the bola-like structure must be folded into an extended, flexible, tertiary structure. These legs, each of about 65,000 Da, appear to be attached near the ends opposite the heads, probably by disulfide bonds. Each leg possesses a tab or protein (approximately 20,000 Da) which juts out from this attachment point.

Amino Acid Sequence↗
PubMed1981

Allogeneic antibody response in cattle.

Antibody responses following immunization with allogeneic bovine lymphocytes, goose red blood cells (GRBC), and ovalbumin (given IV) were studied in 20 heifers. Antibody to GRBC was detectable by 4 to 9 days in all heifers and followed the characteristic pattern of a low-titer primary response which was predominantly immunoglobulin (Ig) M, whereas a high titer IgG secondary response was observed. The dose of ovalbumin used failed to elicit a detectable antibody response. An immune response to allogeneic lymphocytes was observed, but it differed from the GRBC response. Cytotoxic antibody directed against lymphocytes was detectable by day 14 in 5 of the heifers. Five others responded by days 17 to 24 after secondary immunization. The responses were of low titer and predominantly IgM, after both primary and secondary immunizations. The time of antibody appearance and titers varied between heifers and was not dependent on differences at the major histocompatibility complex (BoLA) between heifers and immunizing lymphocytes. Cytotoxic antibody reacted with both B and T lymphocytes and appeared to recognize the serologically defined BoLA antigens. Noncytotoxic alloantibody was detected in the serum of 1 heifer and appeared to be directed against a subpopulation of lymphocytes, possibly those bearing Ia-like antigens.

Animals↗
PubMed1993

An attempt to identify genetic markers of resistance or susceptibility to dermatophilosis in the zebu Brahman population of Martinique.

Dermatophilosis is a disease associated with the tick Amblyomma variegatum, and a genetic predisposition to the manifestation of symptoms has been demonstrated. Indeed, the Creole cattle of Guadeloupe constitute a population which is highly resistant to this disease, whereas the Brahman zebu cattle of Martinique seem very susceptible. However, in this Brahman population there is a gradient regarding the severity of symptoms depending on individuals. In several herds of these pure zebu Brahman, kept under the same farming conditions, we selected susceptible and resistant groups because of the simultaneous presence of animals affected or not by clinical dermatophilosis. In these animals we studied several highly polymorphic genetic systems such as haemoglobin, albumin, the BoLA Complex (class I and II) and the gamma S crystallin gene. Only exon 2 of the BoLA-DRB3 gene, investigated by PCR-RFLP technique, showed interesting polymorphisms. We have established a genotypic map showing at least 4 different alleles of which 1 seems particular to one animal susceptible to dermatophilosis. Before reaching any conclusion further investigations with more DNA samples of susceptible animals are needed.

Actinomycetales Infections↗
PubMed1997

Identification and expression of uvi31+, a UV-inducible gene from Schizosaccharomyces pombe.

The Schizosaccharomyces pombe uvi31+ gene has been previously isolated as a UV-inducible gene [Lee JK et al. (1994) Biochem Biophys Res Commun 202:1113-1119]. This gene encodes a protein of about 12 kDa with 57% amino acid sequence similarity to Escherichia coli BolA protein which is known to be involved in switching between the cell elongation and septation systems during the cell division cycle. The putative Mlul cell cycle box (MCB), SWI4/6-dependent cell cycle box (SCB), and gear-box elements are found in the upstream region of uvi31+ gene, suggesting that this gene shows the cell cycle-regulated and growth phase-dependent expression. Interestingly, the level of uvi31+ transcript varies throughout the cell cycle, peaking in G1 phase before septation, and also shows the growth phase-dependent pattern during cellular growth, increasing maximally at the diauxic shift phase just before stationary phase. Furthermore, the transcript level of this gene is raised after S phase arrest, and is also increased maximally at 4 hr after UV irradiation of 240 J/m2. These results suggest that the delayed induction of uvi31+ gene after UV irradiation may be caused by cell cycle control of this gene after DNA replication checkpoint arrest. Thus, the uvi31+ gene may play a role in controlling the progress of the cell cycle after DNA damage (UV irradiation).

Amino Acid Sequence↗
PubMed2000

Giant vesicles from 72-membered macrocyclic archaeal phospholipid analogues: initiation of vesicle formation by molecular recognition between membrane components

Stereochemically pure archeal acyclic bola-amphiphilic diphosphates 4 and 5, with the basic structure of the phospholipids found in Sulfolobus, have been synthesized for the first time. The self-assembly properties have been compared with those of the nearly identical 72-membered macrocyclic tetraether phosphates 3a and 3b, analogues of the major phospholipid components of Sulfolobus, Thermoplasma, and methanogenic Archea, which were also synthesized. Phase contrast and fluorescence microscopies have shown that the dipolar lipids 1 and 2 spontaneously formed vesicles. Whereas the macrocyclic dipolar phosphates 3 spontaneously formed vesicles (phase contrast and fluorescence microscopies), the bolaform phosphate 4 gave only a lamellar structure (synchrotron diffraction pattern: repeat distance of about 4.25 nm but with only a few layers). However, upon addition of the unphosphorylated precursors phytanol, phytol, or geranylgeraniol to the acyclic lipids 4 and 5, giant vesicles were rapidly formed. Addition of n-hexadecanol or cholesterol did not lead to vesicle formation. Therefore it was concluded that this vesicle formation occurs only when the added molecule is closely compatible with the constituents of the lipid layer and can be inserted into the double layer. A slight mismatch (cholesterol or n-hexadecanol/polyprenyl chains) is therefore enough to block the insertion process presumably required for vesicle formation.

Journal Article↗
PubMed2002

Regioselective photolabeling of glycoprotein A in membranes.

We have developed a chemical method for directly identifying the amino acid residues of the transmembrane domain of a protein that are located right in the center of the membrane. Glycophorin A (GPA), the major sialoglycoprotein of human erythrocytes, was the first membrane protein whose primary sequence was elucidated, but its three-dimensional structure is still not known. GPA has been reconstituted into liposomes formed from dimyristoylphosphatidylcholine, dimyristoylphosphatidylserine, cholesterol, and a bola-amphiphilic phospholipidic photoactivatable probe (radioactive probe 1) by a detergent-mediated method. Electron microscopy confirmed the formation of spherical vesicular structures, and sucrose-density gradients revealed that the proteoliposomes comprised only one membrane fraction. Proteinase-K digestion of GPA in the proteoliposomes suggested that the orientation of GPA in reconstituted proteoliposomes was virtually identical to that observed in natural erythrocyte membranes. After photo-irradiation of the reconstituted proteoliposomes and in situ tryptic digestion, the photolabeled amino acid residues were analyzed by Edman degradation and their radioactivity was measured. Val80 and Met81, which had been assumed to be located near the center of the transmembrane domain of GPA, were indeed highly selectively photolabeled by probe 1. The new method might be applied to analyze the three-dimensional arrangement of the transmembrane domain of protein complexes that are made up from several subunits.

Amino Acid Sequence↗
PubMed1995

Genetic mapping of F13A to BTA23 by sperm typing: difference in recombination rate between bulls in the DYA-PRL interval.

Our objective was to extend the linkage and comparative maps of BTA23 by determining whether the structural gene for the A subunit of blood coagulation factor XIII (F13A) is linked to BoLA-DYA, a centromeric marker, or the distally located gene encoding prolactin (PRL). Bovine F13A was mapped relative to DYA and PRL in an experiment that examined segregation of alleles in 176 sperm. Genotyping was performed by PCR-RFLP for all loci, following amplification of the haploid genome by primer extension preamplification. F13A was found to be linked to PRL (theta = 0.314 +/- 0.038). The most likely order is DYA-PRL-F13A (odds > 10(4):1). This result demonstrates conservation of synteny between BTA23 and most of HSA6p. Surprisingly, theta DYA-PRL was 0.310 +/- 0.039, 83.4% greater (P < 0.02) than we found for another bull (Van Eijk et al., Mamm. Genome 4: 113, 1993). The difference in recombination rate in the DYA-PRL interval provides further evidence for an unusual recombination hot spot between the bovine Mhc class IIa and class IIb subregions and suggests that bull-specific maps may be necessary for marker-assisted selection.

Animals↗
PubMed2001

Identification of a cytotoxic T-cell epitope on the recombinant nucleocapsid proteins of Rinderpest and Peste des petits ruminants viruses presented as assembled nucleocapsids.

The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells. We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E. coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells. Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c). A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response. The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted. Based on the prediction for H-2(d)-restricted T-cell motifs we tested the lysis of transfected P815 (H-2(d)) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins. We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins. Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope. This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides.

Amino Acid Motifs↗
PubMed1995

Extensive MHC class II DRB3 diversity in African and European cattle.

Genetic diversity at the highly polymorphic BoLA-DRB3 locus was investigated by DNA sequence analyses of 18 African cattle from two breeds representing the two subspecies of cattle, Bos primigenius indicus and Bos primigenius taurus. The polymorphism was compared with that found in a sample of 32 European cattle from four breeds, all classified as B. p. taurus. Particularly extensive genetic diversity was found among African cattle, in which as many as 18 alleles were recognized in this small random sample of animals from two breeds. The observed similarity in allele frequency distribution between the two African populations, N'Dama and Zebu cattle, is consistent with the recent recognition of gene flow between B. p. indicus and B. p. taurus cattle in Africa. A total of 30 DRB3 alleles were documented and as many as 26 of these were classified as major allelic types showing at least five amino acid substitutions compared with other major types. The observation of extensive genetic diversity at MHC loci in cattle, as well as in other farm animals, provides a compelling argument against mating-type preferences as a primary cause in maintaining major histocompatibility complex diversity, since the reproduction of these animals has been controlled by humans for many generations.

Africa↗
PubMed1994

Need for cellular and humoral immune responses in bovines to ensure protection from foot-and-mouth disease virus (FMDV)--a point of view.

The published studies on immunization of experimental animals, cattle, and sheep with synthetic peptides containing the antigenic domains in FMDV structural protein VP1 were analyzed. The results obtained with various FMDV synthetic peptides designed to stimulate the humoral immune response in bovines were compared to the current knowledge on MHC class I and class II, and the properties of the peptide binding grooves in each of them. X-ray crystallography of MHC class I proteins provided the three-dimensional structure of the peptide binding groove and led to the isolation and identification of "self" and viral peptides that naturally associate with the peptide binding grooves of both types of MHC and HLA molecules. The available knowledge of the amino acid motifs in MHC and HLA class I-bound viral peptides priming the CD8+ cytotoxic T cell responses must be coupled with the understanding of the three-dimensional structure of BoLA class I. This would aid in the development of an experimental approach to induce bovine anti-FMDV CD8+ cytotoxic cells to complement the humoral immune response to FMDV, which is currently achieved by a killed virus vaccine and, at the experimental level, by a peptide vaccine. Stimulation of both cellular and humoral immune responses against FMDV in cattle may reduce the risk of disease and virus shedding.

Amino Acid Sequence↗
PubMed1999

Evidence for an additional cattle DQB locus.

Studying the genetic polymorphism of the major histocompatibility complex class II genes in cattle, we identified a sequence (KUH1) which resembles those encoding class II beta chains. The gene was shown to be transcribed in peripheral blood leukocytes. Sequence comparisons, Southern blot, and phylogenetic analyses indicate that (1) KUH1 represents a distinct DQB locus, which we propose to designate BoLA-DQB5, (2) DQB5 constitutes an ancient DQB locus which diverged from a common ancestor gene prior to the duplication resulting in DQB1 and DQB2, (3) DQB5 is associated with haplotypes which contain DQA5 and a duplicated DQ region.

Alleles↗
PubMed1996

Polymorphism at the bovine tumor necrosis factor alpha locus and assignment to BTA 23.

We have identified four single-strand conformation variants of the bovine tumor necrosis factor alpha gene by analysis of PCR-amplified fragments. The variants are inherited in Mendelian fashion and are informative for linkage mapping. We have mapped the bovine gene to Chromosome (Chr) 23 in a panel of somatic cell hybrids and observed genetic linkage to the major histocompatibility complex (BoLA) genes and microsatellite markers on bovine Chr 23 in an international bovine reference family panel. The distribution of the alleles was determined in cattle of different breeds and of different geographical origins, which included trypano-susceptible and trypano-tolerant cattle.

Animals↗