Mutations in the gene for the E1β subunit: a novel cause of pyruvate dehydrogenase deficiency

We describe two unrelated patients with pyruvate dehydrogenase (PDH) deficiency attributable to mutations in the gene encoding the E1 subunit of the complex. This is a previously unrecognised form of PDH deficiency, which most commonly results from mutations in the X-linked gene for the E1 subunit. Both patients had reduced immunoreactive E1 protein and both had missense mutations in the E1 gene. Activity of the PDH complex was restored in cultured fibroblasts from both patients by transfection and expression of the normal E1 coding sequence.     

Identification of a mutation within the structural gene for the α subunit of DNA-dependent RNA polymerase of E. Coli

Summary An E. coli mutant rpoA109 unable to support the growth of phage P2 produces DNA-dependent RNA polymerase with an altered subunit. Histidine is substituted for leucine in one tryptic peptide from the mutant subunit. The existence of only one rpoA gene within the E. coli chromosome is indicated.     

Characterization of a G protein α subunit encoded gene from the dimorphic fungus-Tremella fuciformis

Antonie van Leeuwenhoek - Tremella fuciformis is a dimorphic fungus which can undertake the reversible transition between yeast and pseudohypha forms. G protein α subunit (Gα) carries...     

Inactivation of the gene coding for the 30.4-kDa subunit of respiratory chain NADH dehydrogenase: is the enzyme essential for Neurospora?

We have isolated and characterised the nuclear gene that codes for the 30.4-kDa subunit of the peripheral arm of complex I from Neurospora crassa. The single-copy gene was localised on chromosome VI of the fungal genome by restriction fragment length polymorphism mapping. An extra copy of the gene was introduced into a strain of N. crassa by transformation. This strain was crossed with another strain in order to inactivate, by repeat-induced point mutations, both copies of the duplication carried by the parental transformant. Ascospore progeny from the cross were analysed and a mutant strain lacking the 30.4-kDa protein, nuo30.4, was isolated and further characterised. The mutant appears to assemble the membrane arm of complex I, while formation of the peripheral arm is prevented. Nevertheless, the mutant grows reasonably well – indicating that this well conserved protein is not essential for vegetative growth – and is able to mate with other strains both as male or female. Strains with multiple mutations are readily obtained from heterozygous crosses between different complex I mutants of N. crassa. On the other hand, homozygous crosses between several mutants, including nuo30.4, fail to produce ascospores. These results suggest that complex I plays an essential role during the sexual phase of the life cycle of the fungus. Received: 24 February 1997 / Accepted: 23 September 1997     

Comparative genomic analysis of the proteasome β5t subunit gene: implications for the origin and evolution of thymoproteasomes

The thymoproteasome is a recently discovered, specialized form of 20S proteasomes expressed exclusively in the thymic cortex. Although the precise molecular mechanism by which the thymoproteasome exerts its function remains to be elucidated, accumulating evidence indicates that it plays a crucial role in positive selection of T cells. In the present study, we analyzed the evolution of the β5t subunit, a β-type catalytic subunit uniquely present in thymoproteasomes. The gene coding for the β5t subunit, designated PSMB11, was identified in the cartilaginous fish, the most divergent group of jawed vertebrates compared to the other jawed vertebrates, but not in jawless vertebrates or invertebrates. Interestingly, teleost fish have two copies of apparently functional PSMB11 genes, designated PSMB11a and PSMB11b, that encode β5t subunits with distinct amino acids in the S1 pocket. BLAST searches of genome databases suggest that birds such as chickens, turkey, and zebra finch lost the PSMB11 gene, and have neither thymoproteasomes nor immunoproteasomes. In mammals, reptiles, amphibians, and teleost fishes, the PSMB11 gene (the PSMB11a gene in teleost fish) is located next to the PSMB5 gene coding for the β5 subunit of the standard 20S proteasome, indicating that the PSMB11 gene arose by tandem duplication from the evolutionarily more ancient PSMB5 gene. The general absence of introns in PSMB11 and an unusual exon–intron structure of jawed vertebrate PSMB5 suggest that PSMB5 lost introns and duplicated in tandem in a common ancestor of jawed vertebrates, with PSMB5 subsequently gaining two introns and PSMB11 remaining intronless.     

αB-crystallin is a sensor for assembly intermediates and for the subunit topology of desmin intermediate filaments

Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for desmin on CRYAB have been determined, desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of desmin filaments, we show that the binding of CRYAB to desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of desmin-CRYAB myopathies.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Structural analysis and chromosomal localization of the mouse Psmb5 gene coding for the constitutively expressed β-type proteasome subunit

 The proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex class I molecules. Accumulated evidence indicates that, upon stimulation with interferon-γ (IFN-γ), three β-type subunits, designated LMP2, LMP7, and PSMB10, are incorporated into the 20S proteasome by displacing the housekeeping β-type subunits designated PSMB6, PSMB5, and PSMB7, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. In the present study, we determined the organization of the mouse gene Psmb5, coding for the PSMB5 subunit. Psmb5 is made up of three exons, spanning ∼5 kilobases. Its exon-intron organization differs radically from those of the other IFN-γ-regulated, β-type subunit genes including Lmp7 with which Psmb5 is believed to share an immediate common ancestor. The structure of the mouse Psmb5 gene is identical to that of its recently characterized human counterpart. Thus, the unique organization of the gene coding for the PSMB5 subunit appears to have been established before mammalian radiation. As well as the Psmb5 gene, the mouse genome contains a processed pseudogene designated Psmb5-ps. Interspecific backcross mapping showed that Psmb5 maps close to the Gtrgal2 locus on chromosome 14 and that Psmb5-ps is located in the vicinity of the Psme3 locus on chromosome 11. These results were confirmed by fluorescent in situ hybridization analysis that localized Psmb5 to band C2 to proximal D1 of chromosome 14 and Psmb5-ps to band D of chromosome 11. Received: 29 May 1997 / Revised: 4 June 1997     

The isolation of genomic recombinants for the human apolipoprotein B gene and the mapping of three common DNA polymorphisms of the gene —a useful marker for human chromosome 2

Summary We have used four independently isolated cDNA probes for human apolipoprotein B (apo B), to isolate overlapping genomic recombinants for the 3 portion of the apo B gene. The cDNA clones and a unique fragment from the genomic recombinant have been used to identify the human apo B gene in DNA from a series of roden x human somatic cell hybrids. Our results provide evidence for the assignment of this gene to the short arm of human chromosome 2 (p23-pter). We have used the cDNA probes to identify three common DNA polymorphisms. The first, detected with the restriction enzyme XbaI and our probe pAB4, has a rare allele frequency of 0.48. The other two polymorphisms are detected with the probe pAB3. The enzyme MspI detects at least three alleles, with frequencies of 0.67, 0.16 and 0.15, while that detected with the enzyme EcoRI has a rare allele frequency of 0.12. The relative position of these polymorphisms has been mapped using the genomic recombinants.Investigation of a small number of haplotypes indicares that there is linkage equilibrium between the polymorphisms, which have a total polymorphism information content (PIC) value of more than 0.8. These polymorphisms will provide useful markers for genetic studies on chromosome 2 and for the analysis of the involvement of variants of the apo B gene in the development of hyperlipidaemia.     

Construction of a deletion-scanning library for the 5′-proximal promoter region of the bean β-phaseolin gene

A library of internal deletion mutants was constructed in the 5′-proximal promoter region of the gene encoding the bean seed storage protein β-phaseolin. A nick was introduced randomly in the target DNA sequence by depurination and treatment with exonuclease III, and served as the initiating point for deleting adjacent DNA sequences by S1 nuclease. A syntheticPst I linker was ligated to the blunt-ended DNA to serve as a restriction marker for mapping the approximate position of deletion mutants. Subcloning of a kanamycin marker gene into the linker site facilitated selection of plasmid DNA in which internal deletions were introduced in the target DNA sequence. Distribution of internal deletion mutants was mapped by determining the locations of thePst I site in the target sequence. DNA sequence analysis of mutants indicated that the extent of internal deletions ranged from 6 to 100 bp with a mean of 39 bp. In addition, the CAAT and TATA boxes in the promoter region of the β-phaseolin gene were effectively dissected in these mutants.     

Genes for the ‘H’ subunit of human ferritin are present on a number of human chromosomes

Summary DNa has been extracted from hamster-human and mouse-human hybrid cell lines, restricted with EcoRI, and hybridised to a probe for the H subunit of human ferritin, pDBR2. Sequences highly homologous to this probe have been found on at least eight human chromosomes: 1, 2, 3, 6p216cen, 11, 14, 20, and Xq23–25Xqter. Only the gene on chromosome 11 appears to be expressed in these hybrids Southern blotting of DNA from somatic cell hybrids containing different subsets of human chromosomes. The study shows that H subunit sequences are found on at least nine different chromosomes.     

Concept of immunomics: a new frontier in the battle for gene function?

At the beginning of the 21^st century, biology will try to address the function of a large number of new genes. From the perspective of technologies applied today to functional genomics, this task appears to be more complex than the effort invested in the sequencing of the human genome. Conceptually, a high-throughput approach permitting correlation between newly discovered genes and functional properties of their protein products has yet to be developed. To address relationships between tens of thousands of genes and their cognate proteins, novel interdisciplinary technologies need to emerge. In this paper, a new idea of immunomics is presented and an experimental strategy is outlined to circumvent some of the restrictions associated with methodologies currently in use. It is proposed that cloned segments of genomic DNA are used for genetic immunization to obtain a large collection of antibodies, and to generate microarrays of these antibodies for tracing differentially expressed cellular proteins.     

The small subunit ribosomal RNA gene ofTetraselmis convolutae — a feed alga for invertebrate mariculture

This reports the first sequence for a small subunit (185) ribosomal RNA gene (SSU rDNA) fromTetraselmis convolutae.Tetraselmis (=Platymonas) species are important feed stocks for many organisms important in mariculture. This sequence is an essential molecular marker to distinguish the DNAs ofTetraselmis from the DNAs of other organisms in the culture. In addition thisTetraselmis SSU rDNA sequence provides an important entry for the gradually expanding database of symbiotic microalgae.