Class II genes of miniature swine. IV. Characterization and expression of two allelic class II DQB cDNA clones

Two cDNA clones coding for allelic miniature swine MHC class II Ag DQB chains have been isolated, characterized, and shown to be expressed after transfection into mouse fibroblasts. The two alleles differ at the nucleotide level by an overwhelming proportion of replacement substitutions, suggesting the influence of selection for polymorphism. Most of the resulting predicted amino acid replacements are in regions commonly polymorphic in mouse Ab and human DQB sequences, corresponding to the predicted Ag recognition site. Nucleotide and amino acid sequence comparisons to homologous mouse and human sequences show more similarity between swine and man than between either swine and mouse or man and mouse. This tendency is most pronounced when comparing the 3' untranslated regions. However, an examination of unique cross-species sharing of amino acid residues suggests a closer relationship between both man and miniature swine and man and mouse than between miniature swine and mouse. The simplest explanation we can envision for these findings is that the mouse DQB gene homologue (Ab) has been subject to a higher substitution rate than either swine or human DQB genes. An additional cytoplasmic exon expressed in mouse Ab gene products and in putative human DQB2 gene products is lacking in both swine and human DQB cDNA clones. Its absence suggests either that the expression of this exon in mouse Ab genes was activated after mammalian speciation or that the expression of this exon was independently inactivated in swine DQB and human DQB1 genes. Alternatively, the mouse Ab gene may be derived from the same primordial gene as human DQB2, whereas the pig DQB gene may be derived from the same primordial gene as the human DQB1 gene.     

Class II genes of miniature swine

Genomic clones corresponding to class II genes of theSLA ^c haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding theDRB andDQB genes were identified on the basis of hybridization with locus-specific 3 untranslated cDNA probes. Cluster 4 contained exons of bothDOB andDQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containingDP, DR, andDO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the association number M29944. Address correspondence and offprint requests to: C. LeGuern.     

Class II genes of miniature swine

Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.     

Class II genes of miniature swine

Class II genes of miniature swine have been characterized by restriction fragment length polymorphism (RFLP) analysis and by analysis of a series of clones isolated from a lymphocyte genomic library. For RFLP analysis, DNA samples from three independent major histocompatibility complex homozygous lines and three intra-MHC recombinant lines were digested with a variety of restriction enzymes and analyzed in Southern blots using human cDNA probes for DP, DQ, DR, and DZ alpha genes, and DP, DQ, DR, and DO beta genes. One, or at most two, unique fragments were detected by hybridization with each of the human probes tested. In contrast, multiple bands (five to six for most enzymes examined) were detected by each of the human probes tested, the majority of which were found to cross-react with at least three of these probes under conditions of moderate stringency. Genomic DNA from the SLA ^c haplotype was cloned into an EMBL-3 bacteriophage vector, and the corresponding genomic library was screened with each of these human cDNA probes. The class II genes thereby isolated from this library showed characteristics consistent with those anticipated from the RFLP analysis. Thus, unique genes were obtained which showed no evidence of cross-hybridization, while genes showed extensive cross-hybridization and were frequently detected in the library by more than one human gene probe. These data are consistent with early evolutionary divergence of a genes, prior to mammalian speciation, and with continuing evolution of genes, with possible shared usage of these genes by different a loci. The data also imply that genes can readily be assigned to loci homologous to their human counterparts, but that genes will require further mapping and/or sequence analysis to confirm assignments.     

Bovine interferon alpha genes. Structure and expression

The bovine genome contains a gene family of interferon-alpha s (bIFN-alpha) that consists of at least five distinct members. Four of the bIFN-alpha genes isolated show a high degree of homology (97% in the nucleotide sequence and 93% in amino acid sequence). The overall homology in amino acid sequence of bIFN-alpha to human, murine, and rat IFNs-alpha is approximately 60%. Yet there are amino acid clusters (positions 28-41 and 118-146) which are highly conserved throughout the mammalian evolution and in which the overall homology can be as high as 86%. Within the C terminus conserved cluster there is a sequence containing 9 amino acids completely conserved in 16 mammalian IFNs-alpha and of these, 7 are also shared with a similar domain in some bacterial toxins, implying a common functional role for these domains. One of the genes, IFN-alpha C, was expressed in Escherichia coli. The purified bacterial IFN (specific activity, 2 X 10(8) units/mg) exhibited antiviral activity on bovine cells but no detectable activity was demonstrated on human and simian cells.     

Molecular characterization of swine leucocyte antigen class II genes in outbred pig populations

The highly polymorphic swine leucocyte antigen (SLA) genes are among the most important determinants of swine immune responses to disease and vaccines. Accurate and effective SLA genotyping methods are required to understand how SLA gene polymorphisms affect immunity, especially in outbred pigs with diverse genetic backgrounds. In this study, we present a simple and rapid molecular‐based typing system for characterizing SLA class II alleles of the DRB1, DQB1 and DQA loci. This system utilizes a set of 47 sequence‐specific PCR primers developed to differentiate alleles by groups that share similar sequence motifs. We applied this typing method to investigate the SLA class II diversity in four populations of outbred pigs (n = 206) and characterized a total of 19 SLA class II haplotypes, six of which were shared by at least three of the sampled pig populations. We found that Lr‐0.1 (DRB1*01XX–DQB1*01XX–DQA*01XX) was the most prevalent haplotype with a combined frequency of 16.0%, followed by Lr‐0.2 (DRB1*02XX–DQB1*02XX–DQA*02XX) with 14.6% and Lr‐0.15b (DRB1*04XX–DQB1*0202–DQA*02XX) with 14.1%. Over 70% of the pigs (n = 147) had at least one copy of one of these three haplotypes. The PCR‐based typing system described in this study demonstrates a reliable and unambiguous detection method for SLA class II alleles. It will be a valuable tool for studying the influence of SLA diversity on various immunological, pathological and physiological traits in outbred pigs.     

Structure and expression of the genes coding for human alpha 1-acid glycoprotein.

alpha 1-acid glycoprotein (alpha AGP) is a well-characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA segment encoding alpha AGP and we show that it contains three adjacent alpha AGP coding regions, AGP-A, B and B', identical in exon--intron organization but with slightly different coding potential. These results account for the heterogeneity observed by protein sequencing. Southern blot analysis indicates that the cloned cluster contains all the alpha AGP coding sequences present in the human genome. The larger majority of alpha AGP mRNA in human liver is transcribed from AGP-A, whose promoter and cap site have been determined while the level of AGP-B and B' mRNA in human liver is very low. Using Hep3B hepatoma cells as a model system for the in vitro study of the acute phase reaction, we show that only AGP-A is strongly induced by treatment with culture medium of LPS stimulated monocytes.     

Cardiovascular function in anesthetized miniature swine.

Miniature swine anesthetized with pentobarbital were studied with respect to their cardiovascular function under control conditions and in response to catecholamines, baroreceptor inhibition, bilateral vagotomy and vagal nerve stimulation. Measurements included aortic pressure, heart rate, intraventricular pressure and its maximum rate of rise during contraction, carotid blood flow and resistance, femoral blood flow and resistance, and renal blood flow and resistance. The cardiovascular actions of norepinephrine, epiniphrine and isoproterenol were similar to those in other mammals, and the adrenergic receptor mechanisms also were susceptible to blockade with phentolamine or propranolol. Inhibition of the carotid baroreceptors was accompanied by elevation of aortic pressure, reflex bradycardia and increased femoral and renal resistances. Bileteral vagotomy was followed by hypertension, tachycardia and increased renal resistance. Changes in femoral resistance to these procedures differed between the two strains of miniature swine studied. Stimulation of the peripheral end of either vagus nerve was accompanied by bradycardia without hypotension.     

Cardiac function and morphology of Hanford miniature swine and Yucatan miniature and micro swine

The use of miniature swine in biomedical research is increasing; however, a comparison of cardiac function and morphology between strains has yet to be characterized. The purpose of this project was to examine comprehensive hemodynamics and cardiac morphology of three groups of ten normal, 4 months old, age-matched Yucatan miniature (MINI) pigs, Yucatan micropigs (MICRO) and Hanford (HAN) miniature pigs, 5 males and five females per group. Closed chest cardiac catheterization under equivalent conditions was performed followed by post mortem cardiac morphometry. Mean arterial pressure was significantly greater in the Hanford group when compared to both the minipig and micropig pigs (HAN: 89 +/- 4; MINI: 48 +/- 3; MICRO: 53 +/- 2 mmHg). Pulmonary vascular resistance was significantly different between the three groups (HAN: 9 +/- 1; MINI: 60 +/- 12; MICRO: 111 +/- 29 dyne x sec/cm x m2). The Hanford strain had a significantly smaller heart weight to body weight ratio than the other two groups (HAN: 4.6 +/- 1.0; MINI: 5.7 +/- 0.1; MICRO: 5.5 +/- 1.0). Variations in cardiovascular parameters occur among these strains and should be considered when constructing experimental designs.     

Selective expression of class II E alpha d gene in transgenic mice

Class II genes of the MHC must be expressed by APC for activation of CD4+ T cells and efficient delivery of T cell help to B lymphocytes. Class II genes have restricted tissue expression and are under complex regulation. By using various deletion constructs of the class II E alpha d gene in transgenic mice we have mapped different 5' flanking regions which control E alpha d gene expression in distinct cell types. We demonstrate dissociate expression of E alpha d within the macrophage lineage as well as within the B cell lineage, and present evidence for a repressive element operative in B cells and macrophages. We describe the generation of novel transgenic lines with limited constitutive and inducible E alpha mRNA and I-E protein.     

Structure and polymorphism of the HLA class II SB light chain genes

The HLA Class II region contains at least three groups of loci, DR, DC and SB, which play an important role in the immune response. The antigens encoded at these loci are heterodimers composed of an alpha and a beta chain. The sequence of a complete Class II beta cDNA clone whose sequence agrees closely with the limited N-terminal protein sequence available for the SB beta chain is reported. In addition the structure and coding sequence of genomic SB beta clones of two different SB haplotypes has been obtained and allows definition of some polymorphic regions. The SB beta gene appears to undergo alternate splicing at its 3' end, resulting in expression of two different intracytoplasmic regions. Partial sequencing of a second non-allelic SB beta-like gene, SX beta, indicates that it is a pseudogene.     

Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation.

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.     

MHC evolution in three salmonid species: a comparison between class II alpha and beta genes

The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II α and class II β loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II α and β genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIα gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.     

Transplantation in miniature swine. XII. N-terminal sequences of class I histocompatibility antigens (SLA) and beta 2-microglobulin

Purified class I histocompatibility antigens (SLA) from three haplotypes were prepared by papain treatment of lymphoid cell membranes obtained from spleens and lymph nodes of miniature swine homozygous at their major histocompatibility complex. Antigens were purified by ion-exchange chromatography followed by gel filtration. Purity was analyzed by SDS-PAGE, and antigenic specificity by inhibition of complement-dependent, alloantiserum-mediated cytotoxicity. The SLA antigens were reduced and alkylated, and the component heavy and light chains were isolated by gel filtration under dissociating conditions. N-terminal amino acid sequences were obtained for SLAaa, SLAcc, and SLAdd heavy chains, as well as for the light chain, beta 2-microglobulin. The swine antigens showed high levels of homology with class I antigens from other animal species. Heterogeneity was observed among the swine haplotypes, and several of the positions at which substitutions were found are apparently invariant in other animal species. In contrast, only minimal sequence heterogeneity was detected within haplotypes, the basis of which may be of relevance to understanding the evolutionary development of these molecules.