On genetic components in autoimmunity: a critical review based on evolutionarily oriented rationality

The immune system furnishes the organism with the utmost effective defence mechanisms against foreign and changes in self without doing self-harm. However, optimized efficacy in the defence against the immense variety of foreign antigens generates a higher risk for inadvertent self challenge. Such inherent short-comings are the inevitable burden traded for the benefits of an optimally organized defence system. The central molecules involved in specific immune reactions include antigen receptors of B and T lymphocytes, and antigen-presenting proteins encoded by the major histocompatibility complex (MHC; in man HLA). The genetics and evolution of these multigene families is discussed here with respect to their potential contributions to disturbances of self recognition. Simple molecular biological tools and procedures for efficiently screening the immunologically relevant genes are described.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Characterization of hypervariable locus-specific probes derived from a (CAC)5/(GTG)5 multilocus fingerprint in various Eurasian populations

Summary Population genetic studies were performed using oligonucleotide probes (Hz1103, Hz4103, and Hz4201) that recognize three hypervariable loci (D11S859, D9S128 and D22S265) in the human genome. DNA from 17 Eurasian population samples including 37 monozygotic twin pairs were digested with HinfI and hybridized with Hz4103. Allele frequency distribution profiles and high degrees of heterozygosity were similar in each ethnic group. Among 804 unrelated individuals tested, we detected one case of mosaicism caused by a somatic recombination event in a monozygotic twin. In addition, samples of DNA from three ethnic groups (Germans, Assamese Hindus and Thais) and from German and Thai families were restricted with MboI and probed with Hz1103, Hz4103, and Hz4201. The results showed considerable degrees of heterozygosity and locus-specific allele distribution profiles, rather than interpopulation differences. Among 262 meioses (12 three-generation families with a total of 131 children) analyzed, a single recombination event was observed following hybridization with the DNA probe Hz4201.     

Human DNA sequences isolated with an immunoglobulin switch region probe: sequence,chromosomal localization,and restriction fragment length polymorphisms

Summary We have screened a human genomic DNA library with an immunoglobulin (Ig) derived switch (S) region specific probe for homologous sequences. Five Ig independent phage clones were isolated and characterized. The S sequence homologous DNA fragments are short compared to the S region sequences. Ig independent S sequences are flanked by highly repetitive DNA elements and perfect inverted repeats can be demonstrated in their close vicinity. Using subclones of S homologous sequences restriction fragment length polymorphisms were shown within DNA of different T cell leukemias. Burkitt lyphhomas, lymphoblastoid cell lines, and DNA of healthy individuals. One of the five clones isolated with the S region probe was evidently localized to chromosome 2 and/or 40 and showed a complex hybridisation pattern with several different human DNAs. S homologous sequences of another clone are most likely localized on chromosome 1. It is possible that these Ig indenpendent S sequences have arisen by amplification and transposition and that they are involved in genetic recombination.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Geographic differences in the allele frequencies of the human Y-linked tetranucleotide polymorphism DYS19

We have studied the allele frequency distribution of the microsatellite locus DYS 19 in several populations with different geographical origins worldwide. Three new alleles were found. In addition, remarkable geographic and ethnic differences were observed in the allele frequency profiles and DNA marker (gene) diversity among populations and major ethnic groups. Amerindians showed an overwhelming predominance of the A allele, while in Caucasians the B allele was modal, and in Greater Asians and Africans allele C became predominant. Even within these geographic regions there were significant gradients, as exemplified by the decreasing frequency profile of the B allele from Great Britain over Germany to Slovakia. Thus, DYS 19 emerges as a useful tool for studying the structure and dynamics of human populations.     

Chromosomal assignment of the human smg GDP dissociation stimulator gene to human chromosome 4q21-q25

The 3-end of the cDNA encoding the smg GDP dissociation stimulator (smg GDS) protein shares 100% homology with the previously published expressed sequence tag 00038 site. This site extends the 3-end of the smg GDS gene by 212 bp. It has been localized to human chromosome 4. Here, we have refined the localization of smg GDP to human chromosome 4q21-q25 using a mapping panel of rodent/human somatic cell hybrids containing different parts of chromosome 4. This chromosomal localization of smg GDP to 4q21-25 overlaps with a region of allele loss in primary hepatocellular carcinoma (4q13-q26).HGM symbol: RAP1GDS1     

Multilocus DNA fingerprints in the plant Cynoglossum officinale L. and their use in the estimation of selfing

We have developed a nonradioactive oligonucleotide multilocus DNA fingerprinting method for Cynoglossum officinale . Of the 19 probes tested, six probes yielded banding patterns for all restriction enzymes used. All but one of the informative probes are repeats with a four-base motif. Approximately 60% of the loci appeared to be polymorphic. The sensitivity of the nonradioactive method was equal to that of the radioactive method. In addition, a new simple calculation method is presented to estimate selfing rates and approximate 95% confidence limits from the DNA fingerprint profiles avoiding 'between-gel' comparisons. The selfing rates differed significantly (as determined from 95% confidence intervals) between naturally pollinated individuals of C. officinale within the experimental population. The estimates ranged from 0 to 70% selfing.     

Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell

Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold.