Exon-skipping is responsible for the 9 amino acid residue deletion occurring near the N-terminal of human beta-casein.

Interspecies comparison and alignment of the beta-casein N-terminal sequence, taking into account its exon modular splitting derived from the known structural organization of the relevant genes, has revealed that a 9 amino acid residue sequence, corresponding to that encoded by the third exon of the other species genes, is lacking in human beta-casein. Using the polymerase chain reaction technique, we have amplified a human genomic 1-kb fragment, spanning from exon 2 to exon 4, which was subsequently cloned and sequenced. One hundred base pairs (bp) upstream from exon 4 and 737 bp downstream of exon 2, a 27-bp virtual exon 3 sequence, probably skipped during the course of pre-mRNA splicing, was identified. We discuss the possibility that this out-splicing event might be due to the weak strength of the 3' acceptor site and/or to the secondary structure sequestering of the branch site sequence.     

A deletion of the paracellin-1 gene is responsible for renal tubular dysplasia in cattle

Various hereditary diseases analogous to particular human heritable diseases have been identified in cattle. Investigation of these cattle diseases will provide useful information regarding the pathogenesis of the corresponding human diseases. Renal tubular dysplasia is an autosomal recessive disease of Japanese black cattle characterized by renal failure and growth retardation. We have previously mapped the locus responsible for the disease within a region on bovine chromosome 1. In the present study, we further typed additional markers in this region and found that a genomic segment of bovine chromosome 1 including the microsatellite marker BMS4009 was deleted in the affected animals. Construction of a physical map covering this region with BAC clones and comparison of the nucleotide sequences of this region between normal and affected animals revealed that a region of 37 kb including exons 1 to 4 of the bovine paracellin-1 gene was deleted in the affected animals. The paracellin-1 gene, which is the causative gene for human renal hypomagnesemia with hypercaciuria and nephrocalcinosis, encodes a tight junction protein of renal epithelial cells. Therefore, we concluded that deletion of the paracellin-1 gene is responsible for renal tubular dysplasia of cattle, and the cattle disease could be a good model for the human disease.     

The pigeon heart 5''-nucleotidase responsible for ischaemia-induced adenosine formation.

The pH-induced reversible dissociation of pigeon liver malic enzyme (EC 1.1.1.40) was studied by combined use of chemical cross-linking and SDS/polyacrylamide-gel electrophoresis. The tetrameric enzyme showed a pH-dependent dissociation in an acidic environment. At pH values above 8.0 most molecules existed as tetramers. The enzyme was gradually dissociated at lower pH. When the pH was below 5.0 most of the enzyme was present as the monomeric forms. Reassociation of the subunits was accomplished by adjusting the pH to neutrality. The dissociation and reassociation were almost instantaneous. No trimer was detected. The pigeon liver malic enzyme was thus shown to have a double-dimer quaternary structure with D2 symmetry. In the presence of substrates, the monomer-dimer-tetramer equilibrium favours the direction of dissociation. Tartronate, an L-malate analogue, was found to be more effective than L-malate in this process. When the monomeric forms were immobilized, the enzyme subunits were found to be fully active in catalysis. A possible arrangement of the four identical subunits of the enzyme molecule is proposed to account for the results obtained in this investigation. The origin of the half-of-the-sites reactivity of pigeon liver malic enzyme is also discussed.     

An oestrogen-producing seminoma responsible for gynaecomastia.

In feminising testicular tumours, oestrogens can be either secreted by the tumour itself or produced by normal Leydig cells in response to paracrine and/or endocrine stimulation by hCG. Typical hormonal Leydig cell tumour patterns include: plasma oestradiol levels > 300 pmol/l on day 3 following an hCG injection, reduced plasma testosterone, and normal plasma hCG and gonadotrophin levels. Except for elevated plasma oestradiol levels, opposite results are observed in seminomas. We report a case of oestrogen-secreting seminoma mimicking a Leydig cell tumour. A 24-year-old Caucasian patient had complained of gynaecomastia for 6 months before admission. Hormonal pattern was typical of Leydig cell tumour. A 1.4 cm tumour was found in the left testis and confirmed on sonography. Considering the likely diagnosis of Leydig cell tumour, the patient was treated by tumourectomy. Surprisingly, pathological examination revealed a pure seminoma. Perifusion experiments showed that the tumour was able to secrete significant amounts of oestradiol. In addition, hCG induced a two-fold increase in oestradiol production from perifused tumour explants. Immunohistochemistry revealed that the tumour was composed of nests of seminoma cells intermingled with lymphoid infiltrates. Tumour cells also expressed aromatase, the hCG/LH receptor and the Leydig cell marker relaxin-like factor, but were betahCG-negative. These results demonstrate that a pure seminoma of the testis is able to synthesise and secrete oestrogens. They also illustrate that the body of proof favouring the diagnosis of feminising Leydig cell tumour of the testis is not rigorously specific.     

The mechanism responsible for reciprocal BrdU-Giemsa staining

Cytological and biochemical experiments were undertaken to elucidate the mechanisms responsible for the reciprocal Giemsa staining of BrdU-substituted and unsubstituted chromosome regions subjected to high or low pH NaH₂PO₄ treatments. These experiments included staining of chromosome preparations with ethidium bromide (EB), acridine orange (AO), or dansyl chloride, digestion of BrdU-substituted and unsubstituted chromatin with pancreatic DNase I, and SDS polyacrylamide gel electrophoresis of the proteins extracted from, and those remaining in isolated, fixed, air-dried nuclei subjected to either NaH₂PO₄ treatment. The collective evidence from this and previous work clearly indicates that, although the staining reactions following the different pH treatments are reciprocal, the mechanisms of induction of the staining effects are not. After the high pH treatment, BrdU-substituted and unsubstituted chromosome regions are palely and intensely stained with Giemsa, respectively. This treatment preferentially solubilizes BrdU-substituted DNA, probably as a result of the photolysis or high temperature hydrolysis of BrdU-DNA. Concomitantly, this treatment selectively denatures the BrdU-DNA. The reduction in the amount of DNA in the BrdU regions leads to a quantitative decrease in Giemsa-dye binding, resulting in pale staining relative to unsubstituted regions. The extraction of BrdU-substituted DNA does not appear to simultaneously extract much chromosomal protein. After the low pH treatment, BrdU-substituted and unsubstituted regions appear intensely and palely stained with Giemsa, respectively. BrdU substitution greatly increases the binding affinity of histone H1 to DNA, and the low pH treatment preferentially extracts the less tightly bound H1 of the unsubstituted chromatin. This extraction of H1 is presumably responsible for the preferential dispersion of unsubstituted DNA outside the boundaries of the chromosome onto the surrounding area of the slide. The unsubstituted chromosome regions subsequently stain relatively palely with Giemsa, because the DNA in these regions is more dispersed than that in the BrdU-substituted regions. The low pH treatment concomitantly denatures the unsubstituted DNA.     

A three-nucleotide deletion in the UL97 open reading frame is responsible for the ganciclovir resistance of a human cytomegalovirus clinical isolate.

Multiple human cytomegalovirus (HCMV) strains frequently coexist in patients with AIDS, and chronic ganciclovir treatment may favor the emergence of ganciclovir-resistant viral mutants. We report the molecular and biochemical characterization of a HCMV ganciclovir-resistant strain (VR3480) previously recovered from a patient with AIDS who was undergoing multiple courses of ganciclovir treatment (G. Gerna, F. Baldanti, M. Zavattoni, A. Sarasini, E. Percivalle, and M. G. Revello, Antiviral Res. 19:333-345, 1992). Ganciclovir resistance of strain VR3480 was related to impaired ability to monophosphorylate the drug, as indicated by the finding that ganciclovir phosphorylation values for VR3480 were 30% of those shown by the HCMV reference strain AD169 in an in vitro activity assay. Sequencing of the UL97 gene of VR3480, which encodes the viral kinase responsible for ganciclovir phosphorylation, showed an in-frame deletion of three nucleotides resulting in the loss of a leucine at position 595 in the polypeptide. Mutant VR3480 UL97 DNA was able to transfer resistance to the AD169 strain in marker rescue experiments. Analysis of virus isolates and blood polymorphonuclear leukocyte samples spanning the 2-year follow-up period of the patient showed that ganciclovir-resistant strain VR3480 arose ex novo during prolonged antiviral treatment and accounted for the majority of virus load circulating in blood during the period of clinical resistance to ganciclovir treatment.     

T-cell receptor Vbeta deletion and Valpha polymorphism are responsible for the resistance of SWR mouse to arthritis induction

 Collagen type II-induced arthritis (CIA) develops in susceptible mouse strains after intradermal injections of type II collagen (CII) in complete Freund's adjuvant (CFA). Susceptibility to CIA in mice is linked to genes of the major histocompatibility complex (MHC). Although the SWR mouse has a susceptible MHC haplotype (H2 ^q ), it is resistant to CIA. SWR exhibits at least two known immunological defects: (1) it contains a germline deletion of about 50% of T-cell receptor (TCR) Vβ-chain gene segments, and (2) SWR is deficient in complement component C5. It has been shown that T cells that express TCRVα11.1 and TCRVβ8.2 play a substantial role in the pathogenesis of arthritis in the DBA/1 mouse (H2 ^q ). We generated SWR transgenic (tg) mice to determine whether the expression of pathogenic Vα11.1 and/or Vβ8.2 transgenes would confer arthritis susceptibility. Arthritis was induced in the SWR TCRαβ tg mice, but not in SWR TCRβ tg mice. To address the role of Vα11.1 in arthritis susceptibility, we examined the allelic polymorphisms of the Tcra-V11-gene subfamily members between the arthritis susceptible DBA/1 mouse and the arthritis-resistant SWR mouse strain. The amino acid sequences of the Vα11.1 alleles differ at two positions (codons 18 and 68). Accordingly, these two amino acid changes are sufficient to allow the production of pathogenic T cells in SWR mice. This is the first demonstration of the association of a particular Tcra-V allele and arthritis susceptibility in mice. Received: 20 November 1998 / Revised: 15 February 1999     

A method for sequence-specific deletion mutagenesis.

We describe a novel procedure for the construction of deletion mutants. Existing exonuclease-based protocols are efficient at producing randomly positioned deletions over large regions of DNA, but are of limited use in targetted mutagenesis due to their inherent sequence-specificity. We have taken advantage of the Exonuclease III-resistant nature of alpha-thio-dNTPs, incorporated into the target DNA template by a primer extension reaction, to generate base-specific alpha-thio-dNTP terminated products. Following removal of the 5' overhanging strands, the products can be cloned to generate a nested set of deletions with single base-pair increments. We demonstrate the utility of this technique by isolating multiple deletions over a 40bp region of the human beta-interferon promoter.     

The C-terminal half of Hsp90 is responsible for its cytoplasmic localization.

With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C-terminal but not the N-terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C-terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto-NLS). When the C-terminal half of Hsp90 was fused with ER containing no or one proto-NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C-terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.     

The X-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies.

Barth syndrome (BTHS) is an X-linked disorder characterized clinically by the associated features of cardiac and skeletal myopathy, short stature, and neutropenia. The clinical manifestations of the disease are, in general, quite variable, but cardiac failure as a consequence of cardiac dilatation and hypertrophy is a constant finding and is the most common cause of death in the first months of life. X-linked cardiomyopathies with clinical manifestations similar to BTHS have been reported, and it has been proposed that they may be allelic. We have recently identified the gene responsible for BTHS, in one of the Xq28 genes, G4.5. In this paper we report the sequence analysis of 11 additional familial cases: 8 were diagnosed as possibly affected with BTHS, and 3 were affected with X-linked dilated cardiomyopathies. Mutations in the G4.5 gene were found in nine of the patients analyzed. The molecular studies have linked together what were formerly considered different conditions and have shown that the G4.5 gene is responsible for BTHS (OMIM 302060), X-linked endocardial fibroelastosis (OMIM 305300), and severe X-linked cardiomyopathy (OMIM 300069). Our results also suggest that very severe phenotypes may be associated with null mutations in the gene, whereas mutations in alternative portions or missense mutations may give a "less severe" phenotype.     

Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American Saddlebred Horse

Laminin 5 is a heterotrimeric basement membrane protein integral to the structure and function of the dermal–epidermal junction. It consists of three glycoprotein subunits: the α3, β3 and γ2 chains, which are encoded by the LAMA3 , LAMB3 and LAMC2 genes respectively. A mutation in any of these genes results in the condition known as hereditary junctional epidermolysis bullosa (JEB). A 6589-bp deletion spanning exons 24–27 was found in the LAMA3 gene in American Saddlebred foals born with the skin-blistering condition epitheliogenesis imperfecta. The deletion confirms that this autosomal recessive condition in the American Saddlebred Horse can indeed be classified as JEB and corresponds to Herlitz JEB in humans. A diagnostic test was developed and nine of 175 randomly selected American Saddlebred foals from the 2007 foal crop were found to be carriers of the mutation (frequency of 0.026).     

Gene responsible for superinfection exclusion of heteroimmune corynebacteriophage.

Wild-type beta and gamma corynebacteriophages are heteroimmune and infect lysogens of each other productively. Unlike their wild-type counterparts, the bin mutants of each phage are excluded in lysogens carrying the heteroimmune phage. The wild-type phages overcome exclusion by means of the bin gene product which appears to act as an antirepressor. When repression is lifted, exclusion of bin mutants is abolished (N. Groman and M. Rabin, J. Virol. 28:28-33, 1978; J. Virol. 36:526-532, 1980). It has not been clear whether the excluding compound is the immune repressor itself or one whose synthesis is positively regulated by repressor. We have isolated beta exclusion mutants (xcl) that as prophage exhibited normal immune repression but no longer excluded gamma-bin mutants. Furthermore, we have shown that an xcl phage with an active immune repressor acted in trans to continue the positive regulation of exclusion by a second xcl+ prophage whose immune repressor was inactivated. From these results it was concluded that there is a gene distinct from the imm gene which is directly or indirectly responsible for exclusion. The xcl gene, mapped in prophage crosses, was located between imm and bin, that is, in the regulatory region of the phage genome. The simplest hypothesis compatible with the established observations is that beta immune repressor regulates the expression of the xcl and bin genes, the former positively and the latter negatively. It is likely that an analogous regulatory model applies to gamma phage since it has already been shown that both beta and gamma have bin alleles.