Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Polymorphism of the tumor necrosis factor beta gene in systemic lupus erythematosus: TNFB-MHC haplotypes

We investigated the Nco I restriction fragment length polymorphism (RFLP) of the tumor necrosis factor beta (TNFB) gene in 173 patients with systemic lupus erythematosus (SLE), 192 unrelated healthy controls, and eleven panel families, all of German origin. The phenotype frequency of the TNFB*1 allele was significantly increased in patients compared to controls (63.6% vs 47.1%, RR = 1.96, p <0.002). The results of a two-point haplotype statistical analysis between TNFB and HLA alleles show that there is linkage disequilibrium between TNFB*1 and HLA-A1, Cw7, B8, DR3, DQ2, and C4A DE. The frequency of TNFB*1 was compared in SLE patients and controls in the presence or absence of each of these alleles. TNFB*1 is increased in patients over controls only in the presence of the mentioned alleles. Therefore, the whole haplotype A1, Cw7, B8, TNFB*1, C4A DE, DR3, DQ2 is increased in patients and it cannot be determined which of the genes carried by this haplotype is responsible for the susceptibility to SLE. In addition, two-locus associations were analyzed in 192 unrelated healthy controls for TNFB and class I alleles typed by serology, and for TNFB and class II alleles typed by polymerase chain reaction/oligonucleotide probes. We found positive linkage disequilibrium between TNFB*1 and the following alleles: HLA-A24, HLA-B8, DRB1*0301, DRB1*1104, DRB1*1302, DQA1*0501, DQB1*0201, DQB1*0604, and DPB1*0101. TNFB*2 is associated with HLA-B7, DRB1*1501, and DQB1*0602.This study was supported by grants from the Federal Ministry of Research and Technology (BMFT/DFVLR, 01 VM 8608/9), the German Academic Exchange Service (DAAD, 322/501/014/0), and SFB (217).This work is part of the doctoral thesis of M. P. Bettinotti.     

The pollen-specific LIM protein PLIM-1 from sunflower binds nucleic acids in vitro

The protein PLIM-1 (formerly SF3) from sunflower is expressed exclusively in mature, free pollen. It contains two LIM domains associated with an acidic C-terminus comprising six copies of the pentapeptide motif (A,T,S) (E,D) TQN. We have expressed the pollen protein as well as some of its mutant forms inEscherichia coli and have used the bacterially produced proteins to study interactions with nucleic acids. Our studies show that the protein binds DNA and RNA in vitro to form large complexes, while mutant polypeptides containing either a single LIM domain or a destabilized first or second LIM domain do not. Although these data suggest that the biological function of PLIM-1 involves interactions with nucleic acids, its role in pollen development remains unclear.     

Beta-2 microglobulin types in mice of wild origin

To determine the distribution of beta-2 microglobulin (B2m) alleles in wild mice we have typed mice derived from natural populations in Europe, North Africa, South America, and East Asia. Mus musculus domesticus mice from Germany, France, Italy, and Peru were all B2m ^a as were most from the United Kingdom. M.m. musculus mice from Denmark and Czechoslovakia, several stocks of M.m. molossinus from Japan, and M.m. castaneus from China, Thailand, and the Philippines were of B2m ^b type. This is consistent with the notion that C57BL/6 may have obtained some of its genes, including B2m, from Eastern mice. A BgII restriction site characteristic of B2m ^b was also found in mice from Czechoslovakia and Japan, confirming that B2m ^b is a naturally occurring allele of B2m. A new type of ₂m ( ₂m^w1) was found in four stocks of M. spretus from Portugal, Spain, and Morocco. This molecule differs in apparent size and charge from the a and b types. ₂m^w2 was found together with ₂ ma in one stock of M.m. domesticus (brevirostris) from Morocco. ₂m^w3 and ₂m^w4 were found in a few M. m. bactrianus from Pakistan. In all cases tested, these new ₂m molecules associate with class I histocompatibility antigens.Abbreviations used in this paper ₂m beta-2 microglobulin - B2m gene for beta-2 microglobulin - IEF isoelectric focusing - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - MHC major histocompatibility complex - T. E. Tris-EDTA buffer     

Apolipoprotein A-IGiessen (Pro143----Arg). A mutant that is defective in activating lecithin:cholesterol acyltransferase

Apolipoprotein A-IGiessen is a variant form of apo A-I that is displaced from the corresponding normal A-I isoforms on isoelectric focusing gels by a single charge unit towards the cathode [Utermann et al. (1982) J. Biol. Chem. 257, 501-507]. Three subjects heterozygous for the variant were detected in one family. The percentage of the total A-I in plasma represented by the A-IGiessen in these subjects ranged over 25-30%. The variant and normal major A-I isoforms from the proband (Y.J.) were purified by preparative isoelectric focusing and cleaved with CNBr. Analytical focusing of CNBr fragments demonstrated a charge difference between CB3Giessen and normal CB3. Sequence analysis of CB3Giessen revealed that a proline existing in normal A-I was replaced by an arginine in the variant A-I at residue 143. The ability of the mutant A-I to activate purified lecithin:cholesterol acyltransferase was determined in vitro. The cofactor activity of [Arg143]apolipoprotein A-I was about 60-70% of that demonstrated by control A-I. Residue 143 is in a putative beta-turn between two of the repeating amphiphilic helices in apolipoprotein A-I and may be a critical determinant of the protein's structure and function.     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168.

A set of nine reference strains bringing convenient markers in the genetic background of Bacillus subtilis Marburg 168 has been prepared to allow rapid mapping of new markers.     

Isolation and characterization of human Lp-B lipoprotein containing apolipoprotein B as the sole apolipoprotein

A sequential immunoaffinity chromatography procedure was developed to isolate from whole normolipidemic human plasma a subpopulation of apoB containing particles (Lp-B) which is virtually free of non apoB protein. The absence of non apoB protein in Lp-B was assessed by enzyme immunoassay against apolipoproteins A-I, A-II, A-IV, E, C-III and (a). Electron microscopy and fractionation of the isolated particles by gel filtration demonstrated that these particles were heterogeneous in size. However, most of them had diameters between 18 and 26 nm. These particles were found to be rich in cholesterol (molar ratio cholesterol/apoB = 2246 +/- 995) poor in triacylglycerol (molar ratio triacylglycerol/apoB = 555 +/- 518) and had a phospholipids/apoB molar ratio of 713 +/- 348. Most of the cholesterol was esterified (66% +/- 5%). Lp-B particles bound to the apoB, E receptor of HeLa cells with a lower affinity than LDL prepared by ultracentrifugation (1.030 kg/l less than d less than 1.053 kg/l). (KD = 18.9 vs 10.5 nmol/l).     

Genetic control of human apolipoprotein E polymorphism: Comparison of one-and two-dimensional techniques of isoprotein analysis

Summary Genetic polymorphism of human apolipoprotein E (apo E) has previously been demonstrated by one-dimensional isoelectric focusing (Utermann et al. 1977b) and by two-dimensional electrophoresis of apolipoproteins (Zannis et al. 1981), but the relationship between the results obtained by these methods remained unclear. We therefore performed comparative phenotyping by one-dimensional and two-dimensional electrophoresis. Apoproteins from very low-density lipoproteins (apo VLDL) prepared by ultracentrifugation or from an apo Erich lipoprotein fraction prepared by heparin/Mg⁺⁺ precipitation, were used as a source of apo E. Six common phenotypes designated apo E-4/4, apo E-N/N, apo E-D/D, apo E-4/N, apo E-4/D, and apo E-N/D were differentiated irrespective of the technique used or the source of apolipoproteins, but the two-dimensional electrophoresis of apo VLDL and apo VLDL which had been treated with neuraminidase was the key for the correct genetic interpretation of those phenotypes exhibiting the E4 isoform of the protein. Each phenotype is characterized by the presence of either one or two of three major isoforms E2, E3, and E4 and by the presence of several minor sialylated forms of these proteins (apo E_s) that have higher apparent molecular weights. The unsialylated major isoform apo E2 does not only differ in charge but also has a higher apparent mol.wt. (about 34,500) than the major isoforms apo E3 and apo E4 (mol. wt. about 33,000). Family studies including 90 matings with a total of 203 offspring confirmed the genetic one locus model of Zannis et al. (1981). Apo E phenotypes are controlled by three autosomal codominant alleles apo E^d, apo Eⁿ, and apo E⁴ that specify for the E2, E3, and E4 isoforms respectively. Phenotypes apo E-D/D,-N/N, and-4/4 represent homozygotes and phenotypes apo E-4/N,-4/D, and-N/D heterozygotes for these alleles.The frequencies of apo E alleles in 1031 blood donors were apo E⁴=0.150, apo Eⁿ=0.773, and apo E^d=0.077. Homozygosity for the allele apo E^d is associated with hyperlipoproteinemia type III. Hence a large number of the population (about 1%) are at risk for this specific lipoprotein disorder that is associated with premature atherosclerosis and xanthomatosis.