Mario DeMarchi

Structural and functional characterization of 20S and 26S proteasomes from bovine brain.

Two proteins were isolated, in a stable form, from bovine brain by ion exchange chromatography, gel filtration and ultracentrifugation on glycerol gradient. They were identified as 20S and 26S proteasomes on the basis of molecular mass, migration velocity on non-denaturing gels, immunoreactivity, multipeptidase activity and the 26S proteasome also for dependence on ATP for the degradation of short peptides and ubiquitinylated proteins. However, the 26S proteasome has some properties not yet described for its counterpart of other tissues and from brain of this and other species. In particular, the ATP concentration required by the 26S proteasome to reach maximal peptidase activity was approximately 40-fold lower than the one required for maximal proteolytic activity on polyubiquitinylated substrates. Moreover, plots of substrate concentration vs. velocity gave a saturation curve for the 26S proteasome only, which, for the trypsin-like and post-glutamyl peptide hydrolase activities fitted the Michaelis-Menten equation, whereas for the chymotrypsin-like activity indicated multibinding site kinetics with positive cooperativity (n = 2.32+/-0.38). As concerns the 20S proteasome, its electrophoretic pattern on native gel revealed a single protein band, a feature, to our knowledge, not yet described for the brain particle of any species.

Molecular evidence of triplication in the haptoglobin Johnson variant gene

SummaryThe protein and gene structure of the Hp Johnson variant (Hp3) were analyzed in two related heterozygous individuals. The molecular weight (23kd) and amino acid composition of Hp3 alpha chain were in agreement with the triplicated structure first suggested by Smithies in 1964. Direct gene analysis by Southern blotting showed a three-fold tandem repeat of the same 1.7 kb DNA segment implicated in the Hp2 gene duplication. On the basis of these data a nine exon model for the Hp3 gene is proposed.

A new restriction fragment length polymorphism in the haptoglobin gene region

SummaryDirect gene analysis of the haptoglobin gene region was carried out by Southern blotting using an Hp cDNA as probe. Two types of polymorphism were observed: one due to intragenic duplication, is characterized by a constant fragment length difference of 1700bp observed with several enzymes and by complete correspondence with the protein molecular weight polymorphism; the second type, due to point mutation, was represented by two additional restriction sites for Eco RI and Pst I, with a frequency comparable to that of other genes. These two mutations segregated together in families, suggesting that the recently described Hp related gene is closely linked to the Hp gene. Moreover, they were completely associated with each other. The evolutionary significance of this finding is discussed.

The G4 gene is duplicated in 44% of human immunoglobulin heavy chain constant region haplotypes

Abstract The structure of the human immunoglobulin heavy chain constant region (IGHC), on chromosome 14q32, comprises nine CH genes and two pseudogenes, all originating from multiple duplication events. Continuing evolution of the region is demonstrated by the finding of various types of duplicated and deleted haplotypes, which together add up to 6%. Here we provide molecular and genetic evidence that the G4 gene is duplicated in 44% of IGHC haplotypes in the Italian population. The duplication spans about 20 kb of genomic DNA and probably originated through unequal crossing over. Refined characterisation of the genomic region downstream from the G4 gene improves our knowledge of the evolutionary history of CH genes.

Multiple levels of analysis of an IGHG4 gene deletion.

SummaryHuman immunoglobulin heavy chain constant region (IGHC) genes constitute a typical multigene family, usually comprising eleven genes on the telomere of chromosome 14 (14q32). In this region, deleted and duplicated haplotypes have been reported to exist with considerable frequency. Their origin is the result of either unequal crossing-over or looping out excision. In this paper, we report the characterization of a new type of deletion, involving the IGHG4 gene, in a subject who also carries a larger deletion of a previously described type on the second chromosome. Employment of several methods (polymerase chain reaction, standard Southern blot, pulsed field gel electrophoresis, serological techniques) to analyze these deleted haplotypes has resulted in a level of accuracy in their characterization that has not been achieved in previous cases. The site of recombination responsible for the IGHG4 deletion was restricted to a 2.5-kb region 3′ of the G4 gene; this rules out any possible involvement of the S regions in the recombination process. The usefulness of the various techniques in the characterization of the deletions is also discussed, together with possible future applications in the field.

Dicentric Y chromosome in a patient with gonadal dysgenesis and seminoma

SummaryA male patient with ambiguous external genitalia developed a seminoma in the left inguinal region; his internal genitalia included a streak gonad on the right and a small uterus.Cytogenetic studies demonstrated a dicentric Y chromosome with unstable behavior during cell division, which resulted in 45,X/46,X,dic(Y)/47,X,dic(Y),dic(Y) mosaicism.Immunogenetic studies allowed the identification of the male-determining H-Y antigen on both leukocytes and red cells of the patient.The significance of these results is discussed with respect to recent data on the genetic control of H-Y antigen.

MOLECULAR ANALYSIS OF A CASE OF IgA2 DEFICIENCY

A family with two members with selective IgA2 deficiency was analysed by direct gene analysis with different probes for the IgCH region. No gross gene deletions or rearrangements were detected. Genetic analysis based on serological and molecular markers did not rule out linkage with the IgCH region. However, a defect of other genes not linked to the Ig heavy chain region and controlling the expression of IgA may be possible as well.

Structural Genetic Alterations in Ig Subclass Deficiencies

Abstract Serological screening for IgG2 and/or IgG4 deficient subjects among 22.000 blood donors revealed a number of cases of isolated or multiple deficiencies, isolated IgG4 deficiency being the most common (0.245%). Southern blot analysis showed that all cases of multiple deficiency and two cases of isolated deficiency (IgG2 and IgG4) were due to deletion of the corresponding genes on both chromosomes. In IgG4-deficient subjects with no evident DNA alteration, characterization of several RFLPs within the IGHC region showed an increased frequency of certain alleles. Genotype analysis demonstrated that the variation was due to a significant increase of homozygotes for the associated alleles These data suggest that minor structural defects within the IGHC region, besides gene deletions, may be one of the prominent causes of Ig subclass deficiencies

Molecular Genetics of Haptoglobin

Abstract Southern blotting studies of the Hp and Hp related (Hpr) genes allowed to establish by RFLP linkage studies that the two genes are close to each other, as confirmed by direct molecular mapping. A nine exon structure of the rare Hp Johnson variant gene was hypothesized, supporting the proposal that it probably originated through unequal homologous crossing-over.

HLA-DR, Gm Allotypes and Sex as Risk Factors for Celiac Disease

The susceptibility to celiac disease (CD) conferred by three independent genetic factors, i.e., HLA-DR, immunoglobulin allotypes, and sex, has been analyzed in a group of 140 Italian children (Table 1).