Jan Engberg

A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development is less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 84–102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84–102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250–555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta‐ and intra‐membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.

Free ribosomal DNA molecules from Tetrahymena pyriformis GL are giant palindromes.

Restriction ondonuclease EcoRI was used to study the structure of the free ribosomal DNA molecules from Tetrahymena pyriformis, strain GL. From the following observations we conclude that the free rDNA molecules from Tetrahymena are giant palindromes‡, each containing two genes for preribosomal RNA arranged in rotational symmetry as inverted repeating sequences. Analyses of the sizes of products of partial or complete digestion and quantitative analyses of the products of complete digestion of uniformly 32P-labeled rDNA yielded an RI endonucleolytic cleavage map which showed that the EcoRI recognition sites are arranged symmetrically about the center of the rDNA molecule. When heat-denatured rDNA was rapidly cooled under conditions in which no renaturation would occur between separated complementary strands of DNA, molecules of half the size of the original rDNA molecule were produced. These were double-stranded DNA molecules as evidenced by their resistance to digestion with S1 nuclease. Moreover, they could be digested with EcoRI to produce fragments of sizes which would be predicted from the assumption that each single strand of the original rDNA molecule had folded back on itself to form a “hair-pin” double-stranded DNA structure. Hybridization experiments between ribosomal RNA and purified rDNA showed that each rDNA molecule contains two genes for rDNA. Hybridization of the isolated EcoRI fragments of rDNA with 25 S or 17 S rRNA suggested that the two structural genes for 17 S rRNA are located near the center of the rDNA molecule and the two genes for 25 S rRNA are found in distal positions.

Autonomous rDNA molecules containing single copies of the ribosomal RNA genes in the macronucleus of Tetrahymena pyriformis

Abstract The DNA containing the genes for rRNA (commonly called rDNA) of Tetrahymena sediments in sucrose density gradients considerably slower than the main part of the DNA when DNA from gently lysed whole cells or isolated nuclei are fractionated by this method. In rDNA purified by CsCl gradient centrifugation about 20% of the DNA (40% of the bases in one strand) consists of sequences homologous to 25S and 17S rRNA as determined by DNA-RNA hybridization. The purified rDNA co-sediments in sucrose gradients with O29 phage DNA (M.W. = 11 × 106). Examination by electron microscopy of the rDNA demonstrates that the molecules are linear with a length of 5.65 ±0.6 μm corresponding to a molecular weight of 11 × 106.

Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D☆

A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively.

Phage-display libraries of murine and human antibody Fab fragments.

We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage.

Extrachromosomal ribosomal RNA genes in Tetrahymena structure and evolution

Abstract The macronuclear ribosomal RNA genes from a number of strains within several species of Tetrahymena have been characterized. Restriction enzyme analysis revealed that individual strains all contained entirely homogeneous populations of extrachromosomal palindromic ribosomal DNA, varying in molecular size from 12 × 10 6 to 14 × 10 6 in different strains. Considering that the evolutionary distance among some of the species is estimated to be of the order of 10 6 years, the rDNA from all the species exhibited a strikingly high similarity in the localization of their restriction sites. Nevertheless, differences both inside and outside the gene region were clearly detectable, showing that the rDNA sequences have diverged in all species. Genetic polymorphism with respect to rDNA structure exists in Tetrahymena , but seems to be rare. In only two out of five species examined ( T. borealis and T. pigmentosa ) interbreeding strains differing in rDNA structure were found. While the differences detected in the T. borealis rDNA were confined to a small size difference located at the non-coding ends of the molecule, several differences were detected in the rDNA from the T. pigmentosa strains. One of the differences was shown to be due to the presence of an intervening sequence within the structural gene for 26 S rRNA in some of the strains. An intervening sequence of similar size located at the same position within the 26 S gene region was found by R-loop mapping in all strains of the species T. thermophila . Restriction enzyme analysis indicates that the rDNA from two other species contains a similar intervening sequence, and we therefore suggest that the size and localization of the intervening sequence is evolutionarily stable. The two intervening sequences examined so far, however, are not identical, as revealed by restriction enzyme mapping.

Metabolic studies of small molecular weight nuclear RNA components in BHK-21 cells

Abstract The metabolism of small molecular weight homodisperse nuclear RNA components (snRNA) has been studied in Baby Hamster Kidney (BHK-21) cells. 1. 1. Actinomycin D in concentrations which totally inhibits the synthesis of rRNA has little or no effect on the synthesis of snRNA. Increasing the concentration of actinomycin shows that the synthesis of the snRNA components K and L is much less sensitive than that of A, C and D. 2. 2. 3′-Deoxyadenosine inhibits the synthesis of snRNA less than that of rRNA but no difference in sensitivity within the snRNA components is observed. 3. 3. Labelling with [ Me - 3 H]methionine shows incorporation into components A, C and D but not into K and L. 4. 4. Cytosine arabinoside used in concentrations which blocked DNA synthesis has no preferential inhibitory effect on the synthesis of the snRNA components. 5. 5. The electrophoretic mobilities on polyacrylamide gels of components K and L are changed as a result of heat treatment. 6. 6. The presence of Mg 2+ during phenol extraction increases the amount of component K. 7. 7. Components L, A, C and D have a metabolic half-life of 5–7 days in logarithmically growing cells.

Comparison of primary and secondary 26S rRNA structures in two Tetrahymena species: evidence for a strong evolutionary and structural constraint in expansion segments

SummaryWe have determined the nucleotide sequence of the 26S large subunit (LSU) rRNA genes for twoTetrahymena species,T. thermophila andT. pyriformis. The inferred rRNA sequences are presented in their most probable secondary structures based on compensatory mutations, energy, and conservation criteria. The majority of the nucleotide changes between the twoTetrahymena LSU rRNAs and the positions of a relatively large deletion and of the processing cleavage sites resulting in the generation of the hidden break are all located within the so-called divergent domains or expansion segments. These are regions within the common core of secondary structure where expansions have taken place during the evolution of the rRNA of higher eukaryotes.The dispensable nature of some of the expansion segments has been taken as evidence of their non-functionality. However, our data show that a considerable selective constraint has operated to presesrve the secondary structure of these segments. Especially in the case of the D2 and D8 segments, the presence of a considerable number of compensatory base changes suggests that the secondary structure of these regions is of functional importance. Alternatively, these expansion segments may have maintained characteristic folding patterns because only such structures are being tolerated within otherwise functionally important regions.

Recovery and analysis of human genetic material from mummified tissue and bone

Abstract Using sensitive techniques of molecular biology, we have been able to demonstrate the presence of genomic material of human origin in samples of mummified human tissue and bone from selected archaeological sites in Greenland. This result has far-reaching consequences for both evolutionary and archaeological studies of past human populations.