DM Genes Research Articles

Polymorphism of the HLA-DMA and DMB genes in Rheumatoid Arthritis

To determine whether the HLA-DMA and DMB genes, whose encoded molecules are involved in HLA class II-restricted antigen presentation, contribute to the genetic susceptibility to rheumatoid arthritis (RA). One hundred ninety-one RA patients, 147 control subjects, and 218 HLA-DRB1 genotype-matched control subjects were oligotyped for DMA and DMB genes. DMA*0103 and DMB*0104 were significantly increased in the RA patients compared with the randomly selected and the matched controls, thus indicating a direct influence of the DM genes. After stratification of the patients and matched controls according to DRB1 genotypes, only DMA*0103 was increased in the RA patients with DRB1*01, as well as in the patients negative for the RA-susceptibility DR alleles. Our results suggest that DMA*0103 could play an additional role in the genetic susceptibility to RA.

Presentation of abundant endogenous class II DR-restricted antigens by DM-negative B cell lines.

Peptides derived from endogenous and exogenous antigens compete for binding and presentation via class II molecules. Studies with mutant B cell lines defective in exogenous antigen presentation suggest that HLA-DM molecules facilitate the interaction of foreign peptides and class II molecules. In contrast, presentation of self antigens is not strictly dependent upon HLA-DM, as demonstrated by the ability of these mutant cells to activate T cells specific for endogenous antigens. Two distinct classes of DM-negative cells, T2 cells generated by in vitro mutagenesis and lines derived from bare lymphocyte syndrome (BLS) patients, were able to present epitopes derived from self proteins. Transfection of DM genes into the mutant cells enhanced the presentation of some, but not all, endogenous antigens, suggesting that formation of select endogenous peptide/class II complexes is not dependent upon DM. The efficiency of endogenous antigen presentation in the absence of DM was also dependent on the mutant antigen-presenting cell studied, as the TxB hybrid T2 presented greater amounts of self peptides compared to cells from BLS patients. Thus, additional genes, aside from DM, may regulate the pathway for endogenous antigen presentation.

Association of DM Genes in Systemic Sclerosis is Secondary to the Association with HLA Genes

The contribution of polymorphism of DMA and DMB alleles to the pathogenesis of Japanese Systemic Sclerosis (SSc) was studied in 55 Japanese SSc patients and 77 normal Japanese subjects using the PCR-RFLP (restriction fragment length polymorphism) method. The allele frequencies of DMB 0101 allele were increased in SSc with diffuse scleroderma (70.0% vs 49.4%, p < 0.05, pc = not significant (NS)) and in SSc with antitopoisomerase I antibody (a-Scl-70), (68.2%, p < 0.05, pc=NS). The phenotype frequencies of DMB 0101 in these subgroups of SSc were increased significantly (95.0%, p = 0.014, pc < 0.05; 95.5%, p = 0.0088, pc < 0.05, respectively). In contrast, DMB 0102 and DMB 0103 alleles tended to decrease in diffuse scleroderma and SSc with a-Scl-70, but the decreases were not significant. Association analysis among DMA, DMB, and DRB1 1502 in Japanese SSc with diffuse scleroderma and SSc with a-Scl-70 indicated that the increase in DMA 0101 was not primary, but reflected an increase in HLA DRB1*1502.

Linkage disequilibrium between HLA class II (DR, DQ, DP) and antigen processing (LMP, TAP, DM) genes of the major histocompatibility complex.

TAP, LMP and DM genes map within the major histocompatibility complex (MHC) class II region between the DQB1 and DPB1 loci, and are involved in the processing of peptides bound to HLA class I or class II molecules. In order to determine the various linkage disequilibria existing between these genes and HLA class II genes, we have analyzed TAP1, TAP2, LMP2, DMA, DMB, DRB1, DQA1, DQB1 and DPB1 polymorphisms in 162 unrelated healthy Caucasian individuals. Many positive or negative associations were observed between alleles at these loci, such as between DR/DQ and TAP2, DM or LMP, between DP and DMB, and between TAP2 and DM, TAP2 and LMP. Conversely, no linkage disequilibrium was detected between some closely related genes (DR/DQ and TAP1, TAP1 and TAP2, LMP2 and DM), in agreement with the existence of recombination hot spots in this region. Other weak linkage disequilibria are likely to exist in this region. These data allow to define some conserved MHC class II haplotypes including HLA class II and TAP, LMP and DM alleles. Furthermore, the knowledge of such linkage disequilibria is of outstanding importance in order to avoid misinterpretation of the data when studying MHC class II associations with autoimmune diseases.

Molecular analysis of irradiation-induced and spontaneous deletion mutants at a disease resistance locus in Lactuca sativa.

The major cluster of disease resistance genes in lettuce (Lactuca sativa) contains at least nine downy mildew resistance genes (Dm) spanning a genetic distance of 20cM and a physical distance of at least 6 Mb. Nine molecular markers that were genetically tightly linked to Dm3 were used to analyze nine independent deletion mutants and construct a map of the region surrounding Dm3. This analysis identified a linear order of deletion breakpoints and markers along the chromosome. There was no evidence for chromosomal rearrangements associated with the deletions. The region is not highly recombinagenic and the deletion breakpoints provided greater genetic resolution than meiotic recombinants. The region contains a mixture of high-and low-copy number sequences; no single-copy sequences were detected. Three markers hybridized to low-copy-number families of sequences that are duplicated predominantly close to Dm3. This was not true for sequences related to the triose-phosphate isomerase gene; these had been shown previously to be linked to Dm3, as well as to two independent clusters of Dm genes, and elsewhere in the genome. Two spontaneous mutants of Dm3 were identified; several markers flanking Dm3 are absent in one of these two mutants. The stability of the Dm3 region was also studied by analyzing the genotypes of diverse related cultivars. The 1.5 Mb region surrounding Dm3 has remained stable through many generations of breeding with and without selection for Dm3 activity.

Nucleotide Sequences and the Molecular Evolution of the DMA and DMB Genes of the Bovine Major Histocompatibility Complex

cDNA clones encoding the bovine major histocompatility complex (MHC) class II DM α- and β-chains were isolated and characterized. The BoLA-DMA cDNA clone, MA7, encoded a primary translated product of 260 amino acids, which included a signal peptide of 26 amino acids and a mature polypeptide of 234 amino acids. The BoLA-DMB cDNA clone, MB6, encoded a primary translated product of 262 amino acids, with a signal peptide of 18 amino acids and a mature polypeptide of 244 amino acids. Comparison of the sequences and construction of a phylogenetic tree revealed that both clones are more closely related to human and mouse DM genes than to genes for conventional bovine class II α- and β-chains. Thus, since the bovine DMA and DMB genes are so different from other class II sequences and show evidence of strong conservation (>70%) among the bovine, mouse and human homologues, it seems likely that each of these cDNA clones encodes a functional product, which might perform an important function, as previously established in studies in mouse and man.

Antigen presentation and assembly by mouse I-Ak class II molecules in human APC containing deleted or mutated HLA DM genes.

The behavior of mouse I-Ak molecules was studied in the human Ag presentation mutants T2 and 9.5.3, which contain deleted or mutated HLA DM genes. HLA class II molecules expressed by these APC are defective in presentation of native Ag and are mostly complexed with class II-associated invariant chain peptides (CLIP). In contrast to human class II molecules, a significant proportion of mouse I-Ak molecules expressed in T2 and 9.5.3 were associated with antigenic peptides, indicating that I-Ak/peptide assembly is possible in the absence of the Dm proteins. Thus, the presentation of determinants derived from hen egg lysozyme (HEL), keyhole limpet hemocyanin, and conalbumin was normal in 9.5.3Ak and a conalbumin determinant was presented normally by T2.Ak. However, the keyhole limpet hemocyanin determinant was not presented by T2.Ak, and HEL46-61 was only presented at a low level by these APC. SDS-stable, dimeric I-Ak molecules were expressed by both T2.Ak and 9.5.3Ak and formed late in their intracellular transport. Presentation of HEL46-61 was partially inhibited by disrupting vacuolar acidification in 9.5.3Ak, consistent with I-Ak/peptide assembly in a post-Golgi endosomal compartment. Accordingly, Dm is not an obligatory requirement for MHC class II/peptide assembly. We propose that Dm influences the displacement of CLIP from recently synthesized class II molecules, a process that is likely to be less critical for I-Ak because of its low affinity for CLIP.

Mutants of downy mildew resistance in Lactuca sativa (lettuce).

As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

Mutant human cells defective in induction of major histocompatibility complex class II genes by interferon gamma.

Using immunoselection, we have isolated 11 independent mutant HT1080 fibrosarcoma cell lines defective in the induction by interferon gamma (IFN-gamma) of the expression of the human leukocyte antigen HLA-DRA. The mutations are recessive and fall into five complementation groups. All the mutants are affected mainly in the expression of major histocompatibility complex class II and invariant-chain genes. Type I mutants (three complementation groups) are completely defective in induction of the invariant-chain and class II HLA-DP, -DQ, -DR, and -DM genes, whereas type II mutants (two complementation groups) induce these genes weakly in response to IFN-gamma, in the order DPB > DRA > invariant chain. The induction by IFN-gamma of the mRNAs for class I, TAP1, LMP7, and 9-27 is partially defective and the induction of the proteins IRF-1 and ICAM-1 is normal in both types of mutants. All the mutants respond normally to IFN-alpha. The mutants are stable and thus can be used to clone the affected genes by reversion.

Temperature and genotype interactions in the expression of host resistance in lettuce downy mildew

The influence of temperature and genotype on infection types determined by 13 gene-for-gene interactions between lettuce and the downy mildew fungus, Bremia lactucae, was evaluated. Lettuce seedlings containing each of 13 resistance ( Dm) genes were inoculated with isolates of B. lactucae expressing various combinations of the matching avirulence genes, and incubated at 5, 10, 15, or 22 °C. Resistance, assessed by the absence of sporulation, became less effective at lower temperatures for Dm6, Dm7, Dm11, Dm15, and Dm16. No change was observed for resistance conditioned by eight other gene-for-gene interactions. Histological examination indicated that the extent of fungal development in host tissue was dependent on the interacting genotypes and on temperature. Manipulation of interactions involving Dm7 and Dm11, using temperature-shift incubation regimes, indicated that continued and cumulative exposure to permissive temperatures was required for effective resistance and suggested that the determinants of specificity are present in most host cells and are expressed throughout fungal development.

The Level of Field Resistance to Bremia lactucae in Lettuce (Lactuca sativa) Cultivars Carrying the Resistance Gene Dm11

AbstractForty‐one lettuce cultivars (butterhead type), carrying race‐specific resistance gene Dm11 in combination with some others, were studied for field resistance to Bremia lactucae. Significant differences in the level of field resistance were revealed. Most of the cultivars under study were highly susceptible under field conditions. There are evident relationships between combinations of race‐specific Dm genes and field resistance. The cultivars with gene combinations Dm2, Dm3 and Dm11 are susceptible. A very high level of field resistance was detected in the cultivars with combination of genes Dm5/8, Dm6 and Dm16 (cvs. ‘Celia’, ‘Katanga’ and ‘Musette’). This is in close link with their Lactuca serriola origin and a high level of field resistance in progenitors.

Identification of restriction fragment length polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce, using near-isogenic lines

Near-isogenic lines were used to identify restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers linked to genes for resistance to downy mildew (Dm) in lettuce. Two pairs of near-isogenic lines that differed for Dm1 plus Dm3 and one pair of near-isogenic lines that differed for Dm11 were used as sources of DNA. Over 500 cDNAs and 212 arbitrary 10-mer oligonucleotide primers were screened for their ability to detect polymorphism between the near-isogenic lines. Four RFLP markers and four RAPD markers were identified as linked to the Dm1 and Dm3 region. Dm1 and Dm3 are members of a cluster of seven Dm genes. Marker CL922 was absolutely linked to Dm15 and Dm16, which are part of this cluster. Six RAPD markers were identified as linked to the Dm11 region. The use of RAPD markers allowed us to increase the density of markers in the two Dm regions in a short time. These regions were previously only sparsely populated with RFLP markers. The rapid screening and identification of tightly linked markers to the target genes demonstrated the potential of RAPD markers for saturating genetic maps.

A multipoint linkage map around the locus for myotonic dystrophy on chromosome 19

Employing 16 polymorphic DNA markers as well as the chromosome 19 centromere heteromorphism, we have performed a genetic linkage study in 26 families with myotonic dystrophy. Fourteen of these markers had been assigned previously to one of five different intervals of the 19cen-19q13.2 segment by using somatic cell hybrids. For the long arm of chromosome 19, a genetic map that encompasses 9 polymorphic markers and the DM gene has been constructed. Our studies indicate that the DM and CKMM genes map distal to the ApoC2-ApoE gene cluster and to the anonymous polymorphic markers D19S15 and D19S16, but proximal to the D19S22 marker. The orientation of DM and CKMM remains to be determined.

Fungal development and irreversible membrane damage in cells of Lactuca sativa undergoing the hypersensitive reaction to the downy mildew fungus Bremia lactucae

A plasmolytic method was developed to examine irreversible membrane damage (IMD) occurring during the early stages of the hypersensitive reaction (HR) of lettuce ( Lactuca sativa L.) to Bremia lactucae Regel. Symptoms associated with incompatibility determined by Dm genes 1–8 in lettuce were compared. The timing of IMD varied according to the Dm gene expressed and occurred within 1·5 h of penetration during the expansion of the primary vesicle in the most rapid reactions which were determined by Dm 2 and Dm 5/8. By contrast, with Dm 1 or Dm 3, IMD occurred only after initiation of the secondary vesicle, from 3 h after penetration, and rarely occurred during the first 12 h after inoculation of genotypes possessing Dm 4, Dm 6 or Dm 7. The genetic background of the host had little effect on the expression of Dm genes 1, 3 or 5/8, but some fungal isolates, thought to have the same gene for avirulence, differed in their behaviour on resistant cultivars. The time of occurrence of IMD in the first 12 h after inoculation had a direct bearing on the extent of fungal development during the subsequent 36 h. In lettuce cv. Valmaine ( Dm 5/8 gene for resistance) growth of the avirulent isolate VO/11 of B. lactucae was checked at the time of IMD. In all the combinations of isolates and cultivars studied, plasmolysis demonstrated lack of adhesion between primary and secondary vesicles and the host plasmalemma during compatible or incompatible interactions. Variation existed between isolates in their speed of germination and initiation of penetration on different varieties, but variation was not related to the expression of Dm genes.

Genetic analysis of factors for resistance to downy mildew (Bremia lactucae) in species of lettuce (Lactuca sativa and L. serriola)

Simultaneous genetic analysis of resistance in species of lettuce (Lactuca sativa and L. serriola) and a virulence in Bremia lactucae were made to characterize the genetic basis of resistance to downy mildew in 11 host cultivars and accessions. Four new downy mildew resistance genes (Dm13, Dm14, Dm15 and Dm16) were characterized. Other resistance phenotypes were conditioned by combinations of these or previously described Dm genes. New resistance genes, Dm 16 (from LSE/18), Dm14 (from Gelber Winterkonig) and Dm15 (from PIVT 1309), mapped to Dm linkage group I (Dm1, Dm2, Dm3 and Dm6) while Dm13 from Pennlake segregated independently of the three previously characterized linkage groups and therefore belongs to a fourth linkage group. Characterization of new resistance genes requires simultaneous consideration of host and pathogen genetics.