Compound heterozygotes for filaggrin gene mutations do not always show severe atopic dermatitis.

BACKGROUND: Mutations in FLG, which encodes profilaggrin, cause ichthyosis vulgaris (IV) and are an important predisposing factor for atopic dermatitis (AD). IV shows autosomal hemidominant (semidominant) inheritance, and patients with bi-allelic FLG mutations tend to have severe IV phenotypes. However, the effect of bi-allelic FLG mutations on AD incidence and severity remains a subject of controversy. OBJECTIVE: In this study, we studied individuals with bi-allelic null FLG mutations to elucidate the effect of bi-allelic FLG mutations on AD incidence and severity. METHODS: Six individuals with bi-allelic FLG null mutations from three families of IV/AD were investigated. We report the detailed clinical features of the individuals. The phenotype was confirmed by the clinical examinations and the severity of IV and AD was scored using ichthyosis score and Eczema Area and Severity Index (EASI). RESULT: It was found that five of the six patients had severe IV, and the remaining patient showed moderate IV. Two of the six had moderate AD and three of the six had mild AD. The remaining patient had no AD. CONCLUSION: Our results suggest that individuals with bi-allelic FLG mutations do not always have severe AD and confirm that not all individuals with bi-allelic FLG mutations have AD.

Mammalian mitochondrial ribosomal proteins. N-terminal amino acid sequencing, characterization, and identification of corresponding gene sequences.

The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.

Identification and characterization of the genes for mitochondrial ribosomal proteins of Saccharomyces cerevisiae.

We have purified 13 large subunit proteins of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae and determined their partial amino acid sequences. To elucidate the structure and function of these proteins, we searched for their genes by comparing our sequence data with those deduced from the genomic nucleotide sequence data of S. cerevisiae and analyzed them. In addition, we searched for the genes encoding proteins whose N-terminal amino acid sequences we have reported previously [Grohmann, L., Graack, H.-R., Kruft, V., Choli, T., Goldschmidt-Reisin, S. & Kitakawa, M. (1991) FEBS Lett. 284, 51-56]. Thus, we were able to identify and characterize 12 new genes for large subunit proteins of the yeast mitochondrial ribosome. Furthermore, we determined the N-terminal amino acid sequences of seven small subunit proteins and subsequently identified the genes for five of them, three of which were found to be new.

A 570-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 28.0-40.1 min region on the linkage map.

The 569,750 base pair sequence corresponding to the 28.0-40.1 min region on the genetic map of Escherichia coli K-12 (W3110) was determined. This region includes the replication terminus region and contained at least 549 potential open reading frames. Among them, 160 (29%) were previously reported, 174 (32%) were homologous to other known genes, 102 (18%) were identical or similar to hypothetical genes registered in databases, and the remaining 113 (21%) did not show a significant similarity to any other gene. Of interest was the finding of a large number of genes and gene clusters in and near the replication termination region which had been thought to be genetically silent. Those included a cluster of genes for fatty acid beta-oxidation, the third copy of the pot (spermidine/putrescine transport system) gene cluster, the second dpp (dipeptide transport system) operon, the second dsm (anaerobic dimethyl sulfoxide reductase) operon, a cluster of fim (fimbrial) genes and a DNA helicase-like gene with a high molecular weight. In addition, we found the dnaC- and dnaT-like genes in the cryptic prophage, Rac, and a number of genes originated probably from plasmids.

A 460-kb DNA sequence of the Escherichia coli K-12 genome corresponding to the 40.1-50.0 min region on the linkage map.

The 465,813 base pair sequence corresponding to the 40.1-50.0 min region on the genetic map of Escherichia coli K-12 (W3110) was determined. Analysis of the sequence revealed that this region contained at least 466 potential open reading frames, of which 187 (40%) were previously reported, 105 (23%) were homologous to other known genes, 103 (22%) were identical or similar to hypothetical genes registered in databases, and the remaining 71 (15%) did not show a significant similarity to any other gene. At the 45.2-46.0 min region, we found a very large cluster of about 30 genes, whose functions are involved in the biosynthesis of polysaccharides as the components of outer membranes. In addition, we identified a new asn-tRNA gene, designated asnW, between the asnT and asnU genes and a new lysogenic phage attachment site as the cis-element.

Gene MRP-L4, encoding mitochondrial ribosomal protein YmL4, is indispensable for proper non-respiratory cell functions in yeast.

In order to characterize individual protein components of the mitochondrial (mt) ribosome for regulatory, functional and evolutionary studies, the yeast nuclear gene MRP-L4 (accession No. Z30582), coding for the mt ribosomal protein (MRP) YmL4, has been cloned using oligodeoxyribonucleotides (oligos) deduced from a partial amino acid (aa) sequence [Graack et al., FEBS Lett. 242 (1988) 4-8] as screening probes. MRP-L4 is located on chromosome XII and codes for a slightly basic protein of 319 aa. The first 14 aa have not been found in the mature protein, and putatively form a signal peptide that is cleaved off during or after mt import. YmL4 has an N terminus very rich in Pro residues, and at its C terminus contains four hydrophobic domains. YmL4 shows no significant sequence similarity to any other sequence from the databases. Gene disruption shows the MRP-L4 product to be indispensable for mt function in cells growing on non-fermentable carbon sources. In contrast to nearly all other MRPs investigated so far, gene disruption of MRP-L4 also affects growth of yeast cells on fermentable carbon sources, suggesting additional cytosolic and/or mt functions of YmL4 besides its involvement in mt protein biosynthesis.

The yeast nuclear gene MRP-L13 codes for a protein of the large subunit of the mitochondrial ribosome.

The nuclear gene MRP-L13 of Saccharomyces cerevisiae, which codes for the mitochondrial ribosomal protein YmL13, has been cloned and characterized. It is a single-copy gene residing on chromosome XI. Its nucleotide sequence was found to be identical to that of the previously reported ORF YK105. A comparison of the predicted protein sequence of the MRP-L13 gene product and the actual N-terminal amino-acid sequence of the isolated YmL13 protein indicated that the mature protein is preceded by a mitochondrial signal peptide of 86 amino-acid residues, which is the longest among all known mitochondrial ribosomal proteins of S. cerevisiae. No sequence similarity was found to any other ribosomal protein in the current databases. The transcription of MRP-L13 was found to be repressed in the presence of glucose. Its protein product is not strictly essential for mitochondrial functions, but disruption of the gene by insertion of LEU2 noticeably affected cellular growth on non-fermentable carbon sources.

Cloning and nucleotide sequencing of the genes, rpIU and rpmA, for ribosomal proteins L21 and L27 of Escherichia coli.

The rpIU and rpmA genes that encode ribosomal proteins (r-proteins) L21 and L27 of Escherichia coli K-12 have been isolated from the ordered clone bank of this bacterium. They were found to be located at coordinates 3,351.7-3,352.3 kb on the physical map of E. coli. The nucleotide sequence of the cloned genes and their flanking regions indicated that the two r-protein genes compose an operon. Upstream of the two genes there is an open reading frame (ORF) in the opposite direction. The deduced polypeptide encoded by this ORF has a molecular weight of 35,215 and shows a significant degree of sequence similarity to the enzyme that is involved in the carotenoid biosynthesis and encoded by the crtE gene of carotenogenic bacteria and to prenyltransferases found in various organisms.

Efficient large-scale sequencing of the Escherichia coli genome: implementation of a transposon- and PCR-based strategy for the analysis of ordered lambda phage clones.

We have developed a strategy for efficient sequence analysis of the genome of E. coli K-12 using insertions of a Tn5-derived mini-transposon into overlapping ordered lambda phage clones to provide universal primer-binding sites, and PCR amplification of DNA segments adjacent to the insertions. Transposon-containing clones were selected by blue plaque formation on a dnaBamber lacZamber E. coli strain. Insertion points every 0.5-1 kb were identified by 'analytical PCR' and segments between the transposon inserts and phage arms were amplified by 'preparative PCR' using one biotinylated and one non-biotinylated primer. Single strands of amplified DNA fragments were coupled to Streptoavidin-coated paramagnetic beads (Dynabeads M280) through their biotin tails, purified magnetically, and used as templates for fluorescence-based automatic nucleotide sequencing.

YmL9, a nucleus-encoded mitochondrial ribosomal protein of yeast, is homologous to L3 ribosomal proteins from all natural kingdoms and photosynthetic organelles.

The nuclear gene for mitochondrial ribosomal protein YmL9 (MRP-L9) of yeast has been cloned and sequenced. The deduced amino acid sequence characterizes YmL9 as a basic (net charge + 30) protein of 27.5 kDa with a putative signal peptide for mitochondrial import of 19 amino acid residues. The intact MRP-L9 gene is essential for mitochondrial function and is located on chromosome XV or VII. YmL9 shows significant sequence similarities to Escherichia coli ribosomal protein L3 and related proteins from various organisms of all three natural kingdoms as well as photosynthetic organelles (cyanelles). The observed structural conservation is located mostly in the C-terminal half and is independent of the intracellular location of the corresponding genes [Graack, H.-R., Grohmann, L. & Kitakawa, M. (1990) Biol. Chem. Hoppe Seyler 371, 787-788]. YmL9 shows the highest degree of sequence similarity to its eubacterial and cyanelle homologues and is less related to the archaebacterial or eukaryotic cytoplasmic ribosomal proteins. Due to their high sequence similarity to the YmL9 protein two mammalian cytoplasmic ribosomal proteins [MRL3 human and rat; Ou, J.-H., Yen, T. S. B., Wang, Y.-F., Kam, W. K. & Rutter, W. J. (1987) Nucleic Acids Res. 15, 8919-8934] are postulated to be true nucleus-encoded mitochondrial ribosomal proteins.

Cloning and analysis of the nuclear gene for YmL33, a protein of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae.

The N-terminal amino acid sequence of a large subunit protein, termed YmL33, of the mitochondrial ribosome of the yeast Saccharomyces cerevisiae was determined. The data were obtained to synthesize two kinds of oligonucleotide primers, which were used in the polymerase chain reaction to amplify and clone the nuclear gene for this protein. By nucleotide sequencing, the cloned gene, MRP-L33, was found to encode a basic protein of 11 kDa with 98 amino acid residues. The protein encoded by this gene appears to have no leader sequence at its N terminus. The N-terminal two-thirds of the deduced amino acid sequence showed a significant degree of sequence similarity to ribosomal protein L30 of Escherichia coli and Bacillus stearothermophilus. In addition, the C-terminal one-third showed sequence similarity, though to a lesser extent, to a yeast cytoplasmic ribosomal protein termed L16. By hybridization with the yeast chromosomes and their restriction enzyme fragments, the MRP-L33 gene was concluded to exist on chromosome XIII as a single-copy gene. Disruption of the gene by insertion of a HIS3-containing fragment showed that MRP-L33 was essential for mitochondrial function. The transcriptional level of MRP-L33 in strains with different mitochondrial genetic backgrounds was analyzed in the presence of glucose, galactose, or glycerol.

The nuclear coded mitoribosomal proteins YmL27 and YmL31 are both essential for mitochondrial function in yeast.

Using synthetic oligonucleotides deduced from the N-terminal amino acid sequence of purified mitoribosomal protein (mt r-protein) YmL27, the corresponding nuclear gene MRP-L27 of the yeast Saccharomyces cerevisiae has been cloned and sequenced. The MRP-L27 gene codes for 146 amino acids and is located on chromosome X. The mature YmL27 protein consists of 130 amino acids - after cleaving the putative mitochondrial signal peptide - with a net charge of +17 and a calculated relative molecular mass of 14,798 Da. The YmL27 protein as well as the yeast mitoribosomal protein YmL31, which had been characterized and its gene (MRP-L31) cloned previously, is essential for mitochondrial function as shown by the inability of gene disrupted mutants for the MRP-L27 or MRP-L31 genes to grow on non-fermentable carbon sources.

Extended N-terminal sequencing of proteins of the large ribosomal subunit from yeast mitochondria.

We have determined the N-termini of 26 proteins of the large ribosomal subunit from yeast mitochondria by direct amino acid micro-sequencing. The N-terminal sequences of proteins YmL33 and YmL38 showed a significant similarity to eubacterial ribosomal (r-) proteins L30 and L14, respectively. In addition, several proteins could be assigned to their corresponding yeast nuclear genes. Based on a comparison of the protein sequences deduced from the corresponding DNA regions with the N-termini of the mature proteins, the putative leader peptides responsible for mitochondrial matrix-targeting were compiled. In most leader sequences a relative abundance of aromatic amino acids, preferentially phenylalanine, was found.

Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.

Cloning and analysis of the nuclear genes for two mitochondrial ribosomal proteins in yeast.

Two mitochondrial ribosomal proteins of yeast (Saccharomyces cerevisiae) were purified and their N-terminal amino acid sequences determined. The sequence data were used for the synthesis of oligonucleotide probes to clone the corresponding genes. Thus, the genes for two proteins, termed YMR-31 and YMR-44, were cloned and their nucleotide sequences determined. From the nucleotide sequence data, the coding region of the gene for protein YMR-31 was found to be composed of 369 nucleotide pairs. Comparison of the amino acid sequence of protein YMR-31 and the one deduced from the nucleotide sequence of its gene suggests that it contains an octapeptide leader sequence. The calculated molecular weight of protein YMR-31 without the leader sequence is 12,792 dalton. The gene for protein YMR-44 was found to contain a 147 bp intron which contains two sequences conserved among yeast introns. The length of the two exons flanking the intron totals 294 nucleotide pairs which can encode a protein with a calculated molecular weight of 11,476 dalton. The gene for protein YMR-31 is located on chromosome VI, while the gene for protein YMR-44 is located on either chromosome XIII or XVI.

Molecular cloning of the nuclear gene for mitochondrial ribosomal protein YmL31 from Saccharomyces cerevisiae.

The nuclear gene for mitochondrial ribosomal protein YmL31 (MRP-L31) of Saccharomyces cerevisiae was cloned using synthetic oligonucleotide mixtures which correspond to the N-terminal amino acid sequence of the mature YmL31. The gene MRP-L31 codes for a basic protein with a calculated molecular mass of 15.5 kDa and resides on chromosome XI. A comparison of the amino acid sequence deduced from the nucleotide sequence of the MRP-L31 gene and the N-terminal sequence of the isolated protein revealed the existence of a leader peptide sequence of 12 amino acid residues. No significant similarity to known ribosomal protein sequences of other organisms was found.

Characterization of the gene rimK responsible for the addition of glutamic acid residues to the C-terminus of ribosomal protein S6 in Escherichia coli K12.

Ribosomal protein S6 of wild-type strains of Escherichia coli contains up to six glutamic acid residues at its C-terminus. The first two residues are encoded by the structural gene for this protein (rpsF) and the rest are added post-translationally. Mutants deficient in this modification were isolated and characterized genetically and biochemically. The S6 protein in these mutants appeared to contain only two glutamic acid residues at the C-terminus as expected. The mutated gene was termed rimK and was mapped at 18.7 min between cmlA and aroA. The rimK gene was cloned into a cosmid vector and its nucleotide sequence determined. Analysis of the transcriptional and translational products of this gene indicates that it encodes a protein with an Mr of 31.5 kDa and that it forms an operon with a gene encoding a 24 kDa protein. An rpsF mutant containing a Glu to Lys replacement in the second residue from the C-terminus of protein S6 was isolated. The S6 protein of this mutant was apparently inaccessible to the RimK modification system. This indicates that the RimK modification system requires the wild-type amino acid sequence at least in the C-terminal region of ribosomal protein S6.