Iron loading-induced aggregation and reduction of iron incorporation in heteropolymeric ferritin containing a mutant light chain that causes neurodegeneration

Hereditary ferritinopathy (HF) is a neurodegenerative disease characterized by intracellular ferritin inclusion bodies (IBs) and iron accumulation throughout the central nervous system. Ferritin IBs are composed of mutant ferritin light chain as well as wild-type light (Wt-FTL) and heavy chain (FTH1) polypeptides. In vitro studies have shown that the mutant light chain polypeptide p.Phe167SerfsX26 (Mt-FTL) forms soluble ferritin 24-mer homopolymers having a specific structural disruption that explains its functional problems of reduced ability to incorporate iron and aggregation during iron loading. However, because ferritins are usually 24-mer heteropolymers and all three polypeptides are found in IBs, we investigated the properties of Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymeric ferritins. We show here the facile assembly of Mt-FTL and FTH1 subunits into soluble ferritin heteropolymers, but their ability to incorporate iron was significantly reduced relative to Wt-FTL/FTH1 heteropolymers. In addition, Mt-FTL/FTH1 heteropolymers formed aggregates during iron loading, contrasting Wt-FTL/FTH1 heteropolymers and similar to what was seen for Mt-FTL homopolymers. The resulting precipitate contained both Mt-FTL and FTH1 polypeptides as do ferritin IBs in patients with HF. The presence of Mt-FTL subunits in Mt-FTL/Wt-FTL heteropolymers also caused iron loading-induced aggregation relative to Wt-FTL homopolymers, with the precipitate containing Mt- and Wt-FTL polypeptides again paralleling HF. Our data demonstrate that co-assembly with wild-type subunits does not circumvent the functional problems caused by mutant subunits. Furthermore, the functional problems characterized here in heteropolymers that contain mutant subunits parallel those problems previously reported in homopolymers composed exclusively of mutant subunits, which strongly suggests that the structural disruption characterized previously in Mt-FTL homopolymers occurs in a similar manner and to a significant extent in both Mt-FTL/FTH1 and Mt-FTL/Wt-FTL heteropolymers.     

Identification and characterization of polymorphisms at the HAS alpha1-acid glycoprotein (ORM*) gene locus in Caucasians

Human alpha(1)-acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play a crucial role in maintaining homeostasis. Human AGP is the product of a cluster of at least two adjacent genes located on HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locus encoding the AGP genes in a group of healthy Caucasians. Polymorphisms were identified using BamHI, EcoRI, BglII, PvuII, HindIII, TaqI and MspI. Nonrandom associations were found between the BamHI, EcoRI and BglII RFLPs. The RFLPs detected with PvuII, TaqI and MspI were all located in exon 6 of both AGP genes. The duplication of an AGP gene was observed in 11% of the individuals studied and was in linkage disequilibrium with the TaqI RFLP. The identification and characterization of these polymorphisms should prove useful for other population and forensic studies.     

Mucin 4 (MUC4) gene: regional assignment (3q29) and RFLP analysis.

The use of a probe (JER64) containing a mucin 4 (MUC4) cDNA insert of 1.83 kb allowed to assign by in situ hybridization, the MUC4 gene to 3q29. This probe detected RFLPs with all restriction enzymes used (BamHI, HindIII, PstI, EcoRI, and TaqI). Particularly numerous alleles were observed with PstI, EcoRI and TaqI, in a small sample of unrelated DNAs (25 digested with PstI, 8 with EcoRI and 8 with TaqI). The PIC values were 0.69, 0.63 and 0.70 for PstI, EcoRI and TaqI respectively. The polymorphisms observed of variable number of tandem repeat (VNTR) type are in relation with the presence of tandemly repeated nucleotide sequences in MUC4 gene.     

Transforming growth factor-alpha: characterization of the BamHI, RsaI, and TaqI polymorphic regions.

We have characterized the nature of structural alleles of the transforming growth factor-alpha (TGF alpha) locus by restriction-enzyme digestion with BamHI, RsaI, and TaqI. The BamHI polymorphic site is located within exon VI, which codes for the 3' untranslated region. The two BamHI alleles differ by a single point mutation at the restriction site. The RsaI and TaqI polymorphic sites are located within intron V. The two alleles differ at the restriction site, either by a point mutation (RsaI) or by a 4-bp deletion (TaqI). This analysis permits us to devise a PCR method coupled with restriction digestions to directly identify the TGF alpha polymorphisms. Analysis of 99 Caucasian controls has revealed a highly significant (P < .001) association between the RsaI and the BamHI genotype. The frequency of the rare BamHI allele was significantly higher (P < .001) in transformed cell lines (.30) than in controls (.076).     

The use of highly expressed FTH1 as carrier protein for cytosolic targeting in Hansenula polymorpha

The iron storage protein ferritin is a member of the non-heme iron protein family. It can store and release iron, therefore it prevents the cell from damage caused by iron-dioxygen reactions as well as it provides iron for biological processing. To study whether the human ferritin heavy chain (FTH1) can be expressed in Hansenula polymorpha, we integrated an expression cassette for FTH1 and analyzed the protein expression. We found very efficient expression of FTH1 and obtained yields up to 1.9 g/L under non-optimized conditions. Based on this result we designed a FTH1-PTH fusion protein to successfully express the parathyroid hormone fragment 1-34 (PTH) for the first time intracellular in H. polymorpha.     

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.     

Native nonmuscle myosin II stability and light chain binding in Drosophila melanogaster

Native nonmuscle myosin IIs play essential roles in cellular and developmental processes throughout phylogeny. Individual motor molecules consist of a heterohexameric complex of three polypeptides which, when properly assembled, are capable of force generation. Here, we more completely characterize the properties, relationships and associations that each subunit has with one another in Drosophila melanogaster. All three native nonmuscle myosin II polypeptide subunits are expressed in close to constant stoichiometry to each other throughout development. We find that the stability of two subunits, the heavy chain and the regulatory light chain, depend on one another whereas the stability of the third subunit, the essential light chain, does not depend on either the heavy chain or regulatory light chain. We demonstrate that heavy chain aggregates, which form when regulatory light chain is lacking, associate with the essential light chain in vivo-thus showing that regulatory light chain association is required for heavy chain solubility. By immunodepletion we find that the majority of both light chains are associated with the nonmuscle myosin II heavy chain but pools of free light chain and/or light chain bound to other proteins are present. We identify four myosins (myosin II, myosin V, myosin VI and myosin VIIA) and a microtubule-associated protein (asp/Abnormal spindle) as binding partners for the essential light chain (but not the regulatory light chain) through mass spectrometry and co-precipitation. Using an in silico approach we identify six previously uncharacterized genes that contain IQ-motifs and may be essential light chain binding partners.     

Polymorphic class II sequences linked to the rat major histocompatibility complex (RT1) homologous to human DR and DQ sequences

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences.     

Detection and characterization of additional DNA polymorphisms in the dopamine D2 receptor gene.

The gene encoding the dopamine D2 receptor (DRD2) has been suggested as a candidate gene for several mental disorders. We previously described the cloning and chromosomal mapping (to 11q22-q23) of a human DRD2 gene as well as its use for the detection of a two-allele TaqI RFLP with a minor allele frequency of 0.24, corresponding to a PIC of 0.30. Family linkage utilizing DRD2 would be facilitated if the PIC of the DRD2 locus were increased. To this end, we have used additional phage and cosmid clones in the vicinity of DRD2 to identify a new two-allele TaqI RFLP as well as a TG microsatellite polymorphism with a PIC of 0.62. We report localizations of the three polymorphisms on the restriction map of the DRD2 locus. The TaqI RFLPs are in apparent linkage equilibrium with the microsatellite, yielding a highly informative compound marker locus with a PIC of 0.76.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Polymorphism of kappa variable region (V kappa-1) genes in inbred mice: relationship to the Ig kappa-Ef2 serum light chain marker

We describe here the cloning and complete sequence analysis of the rearranged kappa variable region gene from the V kappa-1A-producing myeloma (C.AL20-TEPC-105). A 5'-flanking region probe from the V kappa-1A gene has been used to study the V kappa-1 germ-line gene family in strains of mice differing at the Ig kappa-Ef2 locus. All Ef2a strains examined possess an identical pair of BamHI restriction fragments strongly hybridizing to the 5' probe. Surprisingly, only two of the six strains of mice previously designated Ef2b (NZB and C58) possessed clearly altered restriction fragment sizes for V kappa-1 genes. The remaining four Ef2b strains, namely BDP/J, CE/J, I/LnJ, and P/J, appear to carry V kappa-1 genes similar to those of Ef2a strains. It is suggested that these strains may carry a third form of the V kappa-1A gene, differing in the protein coding region but indistinguishable at the DNA level with the use of BamHI or EcoRI. Alternatively, these strains may fail to express V kappa-1A light chains due to a regulatory defect involving this subgroup of kappa genes.     

DNA polymorphism related to the idiopathic hemochromatosis gene: evidence in a recombinant family

Summary The metabolic error involved in idiopathic hemochromatosis, as well as the underlying genetic defect remain unknown. It has, however, been recently shown that this genetic lesion occurs at a locus linked to the major histocompatibility complex, probably close to the HLA-A locus, and that the disease is recessively transmitted. Therefore, in a family where one subject has idiopathic hemochromatosis his HLA-identical siblings should also be affected. We present here the restriction polymorphism with two MHC class I probes and one DR probe in an exceptional family with three HLA-identical siblings: one (the proband) has a major form of idiopathic hemochromatosis, while the other two are free of any clinical or biochemical signs of the disease. The restriction patterns observed after DNA digestion by enzymes EcoRI, EcoRV, BglII, BamHI, PvuII, TaqI, HincII, and HindIII led to the conclusion that one of the proband's chromosome 6 had undergone two alterations: one, a deletion in the DR region, was revealed by missing fragments all correlated with DR5; the other was an unbalanced cross-over or a genetic conversion in the MHC class I region. This latter alteration was revealed by modifications in the patterns of high molecular weight HindIII bands which hybridize with probe pHLA2 and also by the absence of a HindIII fragment of 7.4 kb hybridized by another class I probe. This latter alteration most likely involved the hemochromatosis gene and could be the first step toward a molecular approach to this gene.     

Somatic cell mapping, polymorphism, and linkage analysis of bovine prolactin-related proteins and placental lactogen.

The bovine prolactin gene family includes novel members expressed in the fetal placenta that are distinct from placental lactogen. In this study, we investigated the genetic organization of four members of this gene family (PRP1, PRP3, PRP6, and PRP10) as well as placental lactogen (PL). Using a bovine-rodent hybrid somatic cell panel, all five genes were assigned to bovine chromosome 23, which contains prolactin and the major histocompatibility group (BOLA). Restriction fragment length polymorphisms were detected by all probes in breeding populations with the restriction enzyme MspI, whereas no polymorphisms were detected with BamHI. EcoRI, HindIII, TaqI, and PstI produced polymorphic fragments with some but not all of the probes tested. A PRP10 polymorphism, which is apparently the result of a insertion/deletion event, detected polymorphism frequency differences between Bos indicus and Bos taurus. No recombinational events were observed with these probes and prolactin using linkage analysis involving 91 American Holsteins. The bovine prolactin gene family was incorporated into a linkage group containing CYP21. Our studies demonstrate that members of the bovine prolactin gene family have a close physical association with each other, and all members demonstrate genetic variability in the breeding population.