Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals

In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR , -DQ , and -DQ cDNA probes in Southern blot analysis. Three subtypes are found using the DR cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, DW10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ and DQ cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ probe, probably due to DX polymorphism. DR RFLP divides the LB 14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases.     

Structural comparison of the genes of two HLA-DR supertypic groups: The loci encoding DRw52 and DRw53 are not truly allelic

The organization and sequence of the HLA-DR chain genes are compared in the two supertypic groups, DRw52 and DRw53, which together account for more than 80% of HLA-DR alleles. From the structural data, we conclude that these two groups represent distinct lineages which have followed different patterns of evolution. The fine structure of the chain locus encoding the DRw53 specificity corresponds most closely to the DR II pseudogene in the DRw52 haplotypes. Concomitantly, the DR I locus in DRw53 haplotypes is more closely related to both of the two expressed DR loci of theDRw5 haplotypes (DR I and DR III). These two loci are the result of a recent duplication. This leads to the proposal that both expressed DR chain genes in the DRw52 haplotypes (DR I and DR III) are derived from a single precursor locus, while the two loci expressed in the DRw53 haplotypes are derived from distinct ancestral loci. The genes encoding DRw52 and DRw53 are therefore not true alleles of the same original locus. A scheme is proposed that accounts for the evolution of DR specificities within the DRw52 and DRw53 groups of haplotypes. It is evident that the differentHLA-DR alleles are not structurally equidistant and that one must take into consideration different degrees of heterozygosity or mismatch among the DR alleles.     

Polymorphism and complexity of HLA-DR: evidence for intra-HLA-DR region crossing-over events

HLA-DR molecules were isolated from HLA-DR3, –5, and –w6 positive homozygous B-cell lines by immunoprecipitation with monoclonal antibodies and analyzed by gel electrophoretic techniques. DNA isolated from the same cell lines was digested with the restriction enzyme Taq I and hybridized with a DR beta full-length cDNA probe. We demonstrated that certain DR I alleles are found in combination with different DR III alleles as defined by Southern blotting, protein chemistry, a functional assay using purified protein derivative-specific T-cell lines, and, in one case, also alloreactive T-cell reagents. Our results indicate that within the family of HLA-DRw52-associated haplotypes DR beta chain genes may have been transferred from one haplotype to another. The implications of these findings are discussed.     

DNA typing of HLA-DR antigens in systemic lupus erythematosus

HLA-DR typing is technically difficult in systemic lupus erythematosus (SLE), where patients have low numbers of peripheral B cells, often of poor viability and weak in antigenic expression. In this series, one third of SLE patients could not be HLA-DR typed by serological techniques, highlighting the potential for systematic bias in DR antigen assignment in studies of HLA and SLE. This potential bias was examined by comparing serological DR results with DNA-DR typing, achieved by examining Taq I fragments of DR, DQ, and DQ which permitted unequivocal DR phenotyping of all patients, including the 35% for whom conventional DR typing was technically impossible. This showed the success of serological DR antigen assignment was indeed nonrandom, with HLA-DR2 and -DR3 significantly more readily identifiable than DR5 and DRw13. Our study suggests that technical problems may well have contributed to conflicting reports regarding the association of DR2 or DR3 with SLE. Here, DNA-DR phenotyping showed HLA-DR3 is significantly (P<.05) associated with an increased risk for SLE (54% in 46 patients, 36% in 134 controls) and HLA-DR2 is not.     

T-cell repertoire in a strain of transgenic C57BL/6 mice with the HLA-DRA gene on the X-chromosome

We have established a strain of transgenic mice in which the HLA-DRA gene was integrated into the X-chromosome and the xenogeneic mixed isotype molecule, DRE^b, was expressed in a cell type-specific manner, although the transgenic DRA gene contained only 268 base pairs of the 5-flanking region. The DRE^b molecules expressed in the transgenic mice functioned as major histocompatibility complex (MHC) class II to select T-cell repertoire, and to stimulate mixed lymphocyte reaction. In female transgenic mice homozygous for HLA-DRA (DR-B6-F-homo) and male transgenic mice (DR-B6-M), DRE^b molecules were expressed in almost all of the MHC class II A^b-positive cells. In contrast, the expression of DRE^b molecules in female transgenic mice hemizygous for HLA-DRA (DR-B6-F-hemi) was found only in part of the A^b positive cells, and the proportion of cells expressing the DRE^b molecules varied due to random inactivation of one of the X-chromosomes. Clonal deletions of the T cells and mature thymocytes bearing Tcrb-V5 and Tcrb-V11, which are eliminated from the peripheral repertoire in mice expressing self-superantigen and MHC class II E molecules, were incomplete in DR-B6-F-hemi as compared with those in DR-B6-F-homo, and were correlated with the proportion of DRE^b-positive spleen cells. These observations suggested that the number of bone marrow-derived cells expressing DRE^b molecules was critical for clonal deletions of Tcrb-V5⁺ and Tcrb-V11⁺ T cells in the thymus.     

MHC class II sequences of an HLA-DR2 narcoleptic

Narcolepsy has a 98% association with the DR2-Dw2/DQw1 haplotype. To establish if a disease-specific allele is present in narcolepsy, a cDNA library was made from a B-cell line from a DR2,4/DQw1,3 narcoleptic. Clones encoding the two expressed DR2 chains, along with DQw1 and chains, were isolated and completely sequenced. The coding regions of these four genes were similar to published nucleotide and protein sequences from corresponding healthy controls, with some minor exceptions. The 3 untranslated region of one of the DR2 genes in the narcoleptic was extended by 42 bp. Complete sequences were not available for DQw1.2 or from healthy individuals, but first domain nucleotide sequences showed only a single nonproductive difference in DQ. Partial protein sequences of both DQ and from published data were identical. Although the effects of minor differences cannot be ruled out completely, it is concluded that there are probably no narcolepsy-specific DR or DQ / sequences, and that the alleles found in narcolepsy are representative of those found in the healthy population.     

GM1 Inhibits Amyloid β-Protein-Induced Cytokine Release

The ganglioside GM1 is known to play a pivotal role in neuronal survival and/or regeneration. Recently it has been shown that GM1 binds tightly with membrane-bound amyloid protein (A) and prevents its conversion from a helical to a -sheet structure. To examine the potential physiological consequences of this binding, we studied the effect of GM1 on A-stimulated release of proinflammatory cytokines, such as interleukin (IL)-1, IL-6 and TNF-, using the human monocytic cell line, THP-1, as a model system. Treatment of THP-1 cells with A 1–40 or A 25–35 resulted in an increased cytokine release from these cells. However, treatment of A-activated THP-1 cells with GM1 and several other complex gangliosides, but not hematosides and neutral glycosphingolipids such as asialo-GM1 (GA1), lactosylceramide, and globoside, significantly decreased the cytokine release. In contrast, this effect was not observed for lipopolysaccharide (LPS)-activated and thrombin-activated THP-1 cells, indicating that the ganglioside effect is specific for A-induced cytokine release. A direct interaction between GM1 and A was demonstrated using the surface plasmon resonance technique. We found that GM1 ganglioside exhibited higher affinity for A 1–40 than GA1, suggesting that the sialic acid moiety of GM1 is necessary for its interaction with A. We conclude that the inhibitory effect of GM1 on A-induced cytokine release may reflect pre-existing abnormalities in membrane transport at the stage of amyloid formation and that GM1 may induce conformational changes in A, resulting in diminished fibrillogenesis and prevention of the inflammatory response of neuronal cells in Alzheimer's disease.     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

A highly divergedβ1 exon in theDR region of the human MHC: Sequence and evolutionary implications

TheDR subregion of the human major histocompatibility complex from aDR4 haplotype includes the well-characterizedDR _ga ,DR4 DR(MT3) andDR genes. In addition, the region between theDR and the proximalDR(MT3) genes contains several copies of conserved DR -related sequences. These repeated elements, numbered II, III, and IV, include the DR signal sequence and a region located further upstream. Further examination of these conserved sequences showed that DR first intron sequences are present at the 3 ends of these repeats. Progressively longer portions of the DR first intron are conserved from repeat II to repeat IV, producing a gradient of conservation. The most complete repeat element of repeats II, III, and IV is associated with a lone1 exon (DR ₁). Upon sequencing, (DR ₁). was found to contain several deleterious mutations, indicating that it is nonfunctional. (DR ₁). has accumulated a large number of replacement substitutions and mutations at positions which are invariant in1 domains from expressedDR genes: 77.8% of the nucleotide substitutions were replacement substitutions, and 41.5 % of the amino acids at invariant positions have been altered. Calculations based on these figures suggest thatDR 1 may have become inactive approximately 25 million years ago. There are, however, two histidine residues within a variable region which are unique toDR 1 and theDR4 gene, suggesting that they represent a gene pair which probably evolved by duplication of a singleDR chain gene.     

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DR gene and an allele, 52a, of locus DR III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DR III locus. The TR81 specificity was also present on the DR I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

An isolated β 1 exon next to the DR α gene in the HLA-D region

A cosmid clone containing the DR gene and a ₁ exon of a DR -related gene was isolated from a human cosmid clone bank made from the consanguineous DR7 cell line MANN. No other DR-related exons were found on this clone. The ₁ exon was located about 15 kb away from the DR gene in a tail-to-tail (3 to 3) orientation. The exon contained several deleterious mutations: a defective splice site at the 5 end, two translational frame shifts (a 1 by deletion and a 1 bp insertion), and three extra cysteine residues. Nucleotide and amino acid sequence comparisons of the ₁ exon indicated that although it is substantially different from other class II -chain genes, it is slightly more related to DR than to any other class 11 gene. The DR-related sequence was on a DNA fragment which showed no polymorphism on a panel of cell lines with Eco RI or Pst 1. These Southern blots, however, revealed a related, polymorphic sequence in the human genome. Nucleotide sequences in the intron flanking the ₁ exon shared greater sequence homology than the ₁ exon itself when compared with the DR genomic sequence. The exon may play a role in the generation of variation in expressed class II -chain genes and it may be a relic of a different subset of class II products.     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta

Summary The restriction fragment patterns of two mutant forms of the temperate Bacillus subtilis bacteriophage SP have been examined. The DNA of a heat-inducible mutant, SPc2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SPc⁺ DNA. The DNA of a clear-plaque mutant, SPc1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SPc1 nor SPc2 DNA is cleaved by the endonuclease HaeIII.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

A study of the HLA-D region in patients with classic Kaposi's sarcoma

The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR and DQ. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group.     

Characterization of an HLA-DR β pseudogene in the DRw52 supertypic group

The nature of the DR II pseudogene in a haplo-type of the DRw52 supertypic group was investigated by nucleotide sequence analysis. It revealed several deleterious mutations in the signal sequence and second domain regions in addition to the complete absence of the first domain and adjacent sequences. No expression of DR II pseudogene mRNA can be detected. The same DR II pseudogene is probably present in other members of the DRw52 supertypic group. The pattern of mutations in this DR II pseudogene is different from that observed in the DR pseudogene of the DRw53 supertypic group, indicating a distinct evolutionary pathway for these two groups of DR haplotypes.     

Evidence for methylation as a regulatory mechanism in HLA-DR x gene expression

We examined the possibility that one mechanism for controlling HLA-DR gene expression occurs through DNA hypomethylation. We employed the restriction enzyme Hpa II, which recognizes the sequence 5CCGG3 but not 5C^mCGG3, to study DNA methylation. We first compared a DR-positive B lymphoblastoid cell line (LCL) with an isogenic DR-negative T-LCL. Using a genomic probe for the DR gene, we showed that an Hpa II digestion of DNA from the B-LCL resulted in bands of lower molecular weight than that of the T-LCL. This indicates that the B-LCL DR gene is hypomethylated relative to the T-LCL gene. Demethylation of the gene from the B-LCL is incomplete, suggesting that complete demethylation is not required for its expression. We also examined somatic cell hybrids of T-LCL and B-LCL since the DR gene, which is inactive in the T-LCL, is expressed in the hybrids, providing a system to study DR gene induction. We examined the hybrid line 174 × CEM.T1, which contains and expresses solely the DR gene from the T-LCL parent since both copies of the DR gene from the B-LCL parent, 174, are deleted. The expressed DR gene from the hybrid was compared with the unexpressed gene from the T-LCL parental line, and again an association between DR gene expression and DNA hypomethylation was observed. In contrast to the DR gene from B-LCL, which is not completely demethylated, the DR gene in this hybrid line is not methylated at either of the Msp I sites covered by our probe. This suggests that different regulatory mechanisms operating through DNA methylation may be involved in the expression of DR genes from T-LCL and B-LCL. Examination of another hybrid line which has DR genes from both parental lines supports this contention. The implications of these findings are discussed.     

Cryptomonad biliproteins — an evolutionary perspective

Each cryptomonad strain contains only a single spectroscopic type of biliprotein. These biliproteins are isolated as 50000 kDa '₂ complexes which carry one bilin on the and three on the subunit. Six different bilins are present on the cryptomonad biliproteins, two of which (phycocyanobilin and phycoerythrobilin) also occur in cyanobacterial and rhodophytan biliproteins, while four are known only in the cryptomonads. The subunit is encoded on the chloroplast genome, whereas the subunits are encoded by a small nuclear multigene family. The subunits of all cryptomonad biliproteins, regardless of spectroscopic type, have highly conserved amino acid sequences, which show > 80% identity with those of rhodophytan phycoerythrin subunits. In contrast, cyanobacteria and red algal chloroplasts each contain several spectroscopically distinct biliproteins organized into macromolecular complexes (phycobilisomes). The data on biliproteins, as well as several other lines of evidence, indicate that the cryptomonad biliprotein antenna system is primitive and antedates that of the cyanobacteria. It is proposed that the gene encoding the cryptomonad biliprotein subunit is the ancestral gene of the gene family encoding cyanobacterial and rhodophytan biliprotein and subunits.Abbreviations Chl chlorophyll - CER chloroplast endoplasmic reticulum - SSU rRNA small subunit ribosomal RNA     

Production of thermotolerant β-amylase byBacillus circulans

An isolate from a Hong Kong soil sample which produced -amylase was identified as a thermotolerant strain ofBacillus circulans with a growth range of 35 to 55C. The -amylase was stable at 45°C for 30 min but lost half of its activity after 30 min at 50°C. Maximum specific activity of -amylase (36.2 units/mg protein) in the culture broth was detected after 36 h of cultivation at 45°C in a medium containing soluble starch, beef extract, coconut water and inorganic salts.