PET1402, a nuclear gene required for proteolytic processing of cytochrome oxidase subunit 2 in yeast

The nuclear mutation pet ts1402 prevents proteolytic processing of the precursor of cytochrome oxidase subunit 2 (cox2) in Saccharomyces cerevisiae. The structural gene PET1402 was isolated by genetic complementation of the temperature-sensitive mutation. DNA sequence analysis identified a 1206-bp open reading frame, which is located 215 by upstream of the PET122 gene. The DNA sequence of PET1402 predicts a hydrophobic, integral membrane protein with four transmembrane segments and a typical mitochondrial targeting sequence. Weak sequence similarity was found to two bacterial proteins of unknown function. Haploid cells containing a null allelle of PET1402 are respiratory deficient.     

Molecular cloning and genetic mapping of the PET494 gene of Saccharomyces cerevisiae

Summary The activity of the nuclear gene PET494 is required to allow expression of the yeast mitochondrial gene oxi2. To aid the study of the mechanism of action of PET494 we have isolated this gene from yeast DNA. A clone bank of yeast DNA fragments in a yeast-E. coli shuttle vector was screened by transformation for a plasmid able to complement the pet494-1 amber mutation. A complementing plasmid was obtained that contained a unique 4.4 kb yeast sequence. This 4.4 kb sequence contains the PET494 gene. Integration of a plasmid containing it into chromosomal DNA by homologous recombination, and subsequent genetic analysis, demonstrated that the 4.4 kb fragment was tightly linked to the pet494-1 mutation. In addition, the corresponding 4.4 kb sequence isolated from a pet494-1 mutant failed to complement the mutation. A 2 kb fragment, subcloned from the original plasmid retained the ability to complement the mutation. The pet494-1 mutation maps to chromosome XIV between rna2 and lys9, approximately 2.4 cm from lys9.     

Mitochondrial inner membrane protease 1 of Saccharomyces cerevisiae shows sequence similarity to the Escherichia coli leader peptidase

Summary The nuclear yeast mutant pet ts2858 is defective in the removal of pre-sequences from the mitochondrially encoded cytochrome oxidase subunit II (COXII) and the processing intermediate of cytochrome b ₂ (Cytb ₂), a nuclear gene product. In order to identify the genetic lesion in this mutant we have cloned and characterized a DNA region which complements the pet ts2858 mutation. The DNA sequence revealed three open reading frames, one of which is responsible for the complementation. A 570 by reading frame represents the structural gene PET2858, as demonstrated by in vitro mutagenesis, gene expression from a foreign promoter, and allelism tests. PET2858 encodes a 21.4 kDa protein, which is essential for growth on non-fermentable carbon sources and for the proteolytic processing of COXII and the Cytb ₂ intermediate. When the N-terminus of the PET2858 protein is fused to a reporter protein, the resulting hybrid molecule is imported into mitochondria. Interestingly, the N-terminal half of the deduced PET2858 protein exhibits 30.7% amino acid identity to the leader peptidase of Escherichia coli. These results suggest that PET2858 codes for a mitochondrial inner membrane protease (IMP1) or at least a subunit of it. This protease is involved in protein processing and export from the mitochondrial matrix.Dedicated to Professor Dr. Peter Starlinger on the occasion of his 60th birthday     

Molecular diagnosis of the transthyretin (TTR) Met111 mutation in familial amyloid cardiomyopathy of Danish origin

Summary Familial amyloid cardiomyopathy in a Danish kindred is associated with a specific mutation (Met for Leu¹¹¹) in the transthyretin (TTR) gene, causing the loss of a recognition site for the restriction enzyme DdeI in the gene. We describe a diagnostic test for the molecular detection of this mutation. A sequence of the TTR gene containing the mutation was amplified by the polymerase chain reaction from isolated genomic DNA of two affected patients and several controls. DdeI digestion of the amplified DNA from the patients revealed 3 bands by gel-electrophoresis, whereas amplified DNA of the controls showed only 2 bands, consistent with complete digestion. Thus, the assumed heterozygous TTR Met¹¹¹ mutation was confirmed in the affected patients.     

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

Summary A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.     

Characterization of Mutations at the Mouse Phenylalanine Hydroxylase Locus

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. InPAH^ENU1,the phenotype is mild. ThePah^enu1mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. InPAH^ENU2,the phenotype is severe. ThePah^enu2mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. InPAH^ENU2,the sequence information was used to devise a direct genotyping system based on the creation of a newAlw26I restriction endonuclease site.     

Nickel subsulfide is genotoxic in vitro but shows no mutagenic potential in respiratory tract tissues of BigBlue™ rats and Muta™Mouse mice in vivo after inhalation

Carcinogenic nickel compounds are known to induce promutagenic DNA lesions such as DNA strand breaks and DNA adducts in cultured mammalian cells. In standard mutation assays, in contrast, they were found to be either inactive or weakly active. In our in vitro mutation studies in a lacI transgenic embryonic fibroblast cell line, nickel subsulfide (Ni₃S₂) increased mutation frequency up to 4.5-fold. We subsequently applied the comet assay and transgenic rodent mutation assays to investigate the DNA damaging effect and mutagenic potential of nickel subsulfide in target cells of carcinogenesis. A 2-h in vitro treatment of freshly isolated mouse nasal mucosa and lung cells with nickel subsulfide clearly induced DNA fragmentation in a concentration dependent manner. The strong effect was not seen in the same cell types following inhalative treatment of mice and rats, leading only in the mouse nasal mucosa to high DNA damage. When the same inhalative treatment was applied to lacZ and lacI transgenic mice and rats, the spontaneous mutation frequency of these target genes in the respiratory tissues was not increased. These results support a recently proposed non-genotoxic model of nickel carcinogenesis, which acts through gene silencing via DNA methylation and chromatin condensation. This model may also explain our in vitro mutation data in the lacI transgenic cell line, in which nickel subsulfide increased mutation frequency, but in about one-third of the mutants, molecular analysis did not reveal any DNA sequence change in the coding region of the lacI gene despite of the phenotypic loss of its function.     

The hha gene modulates haemolysin expression in Escherichia coli

A mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202-312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.     

Molecular tagging and candidate gene analysis of the high gamma-tocopherol trait in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.)

Genetic control of the synthesis of high gamma-tocopherol (gamma-T) content in the seed oil of safflower (Carthamus tinctorius L.) and development of highly reliable molecular markers for this trait were determined through molecular tagging and candidate gene approaches. An F2 population was developed by crossing the high gamma-T natural mutant IASC-1 with the CL-1 line (standard, high alpha-T profile). This population segregated for the partially recessive gene Tph2. Bulked segregant analysis with random amplified polymorphic DNA (RAPD) and microsatellite (SSR) markers revealed linkage of eight RAPD and one SSR marker loci to the Tph2 gene and allowed the construction of a Tph2 linkage map. RAPD fragments closest to the Tph2 gene were transformed into sequence-characterized amplified region markers. A gamma-T methyltransferase (gamma-TMT) locus was found to co-segregate with Tph2. The locus/band was isolated, cloned and sequenced and it was confirmed as a gamma-TMT gene. A longer partial genomic DNA sequence from this gene was obtained. IASC-1 and CL-1 sequence alignment showed one non-synonymous and two synonymous nucleotide mutations. Intron fragment length polymorphism and insertion-deletion markers based on the gamma-TMT sequence diagnostic for the Tph2 mutation were developed and tested across 22 safflower accessions, cultivars, and breeding lines. The results from this study provide strong support for the role of the gamma-TMT gene in determining high gamma-T content in safflower and will assist introgression of thp2 alleles into elite safflower lines to develop varieties with improved tocopherol composition for specific market niches.     

Characterization and disruption of exonuclease genes from <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> B96 and <Emphasis Type="Italic">S. coelicolor</Emphasis> A3(2)

Streptomyces aureofaciens B96 produces several intra- and extracellular enzymes with deoxyribonuclease activity. According to the sequence of the previously published gene exoSc from S. coelicolor A3(2), the DNA sequence from S. aureofaciens B96 was amplified, cloned and expressed in E. coli. The protein product of exoSa gene, recExoSa, was also an exonuclease with DNAase and 5′-phosphomonoesterase activities at optimum temperature 37 °C and pH 8.0. It degraded only linear DNA (chromosomal, double-stranded and single-stranded) and linear plasmid DNA from both ends, with a preference to blunt ends in comparison with overhang ends. The purified enzyme exhibited no RNAase activity. Both exoSc and exoSa genes were interrupted by the apramycin resistance gene; constructed fragments were transformed into particular streptomyces protoplasts. Mutation caused by exoSa disruption in S. aureofaciens chromosome and mutation by interrupted exoSc in S. coelicolor were lethal.     

The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia

The length polymorphism in the waxy gene, which encodes a granule-bound ADP-glucose-glucosyl transferase [granule-bound starch synthase I (GBSS I), E.C. 2.4.1.11] in barley (Hordeum vulgare), was found. The 5′ leader sequence of the waxy gene of barley germplasm from Japan and Korea was analyzed by the polymerase chain reaction (PCR). The waxy gene of these genetic stocks had three types of length polymorphisms, suggesting that there are insertion/deletion mutations at the 5′ leader sequence of the waxy gene. DNA sequence analysis of the polymorphic PCR products showed that: (1) a 403-bp deletion mutation, which included a complete exon I, was found in the wax allele and a 193-bp insertion sequence was located in the intron I, and (2) the insertion sequence was also located in intron I of the Wax allele. The identity of the insertion sequence was completely conserved between the wax allele and the novel Wax allele. These finding s implying that the wax allele, which was found in indigenous waxy barley, originated in non-waxy barley with the novel Wax allele. Received: 12 January 2001 / Accepted: 17 April 2001     

Repair of Double-Strand DNA Gaps in Yeast Saccharomyces cerevisiae: Homology-Dependent Ligation and the Role of the RAD55 Gene

In our previous works, a mutation in the RAD57 gene was shown to induce the plasmid DNA double-strand gap (DSG) repair via a special recombinational repair mechanism: homology-dependent ligation responsible for reuniting disrupted plasmid ends without reconstructing the sequence lost because of the DSG. In this work, the role of the RAD55 gene in the plasmid DNA DSG repair was studied. A cold-sensitiverad55-3 mutation markedly decreased the precision of plasmid DNA DSG repair under conditions of restrictive temperature (23°C): only 5–7% of plasmids can repair DSG, whereas under permissive conditions (36°C), DSGs were repaired in approximately 50% of the cells. In the cold-sensitive mutation rad57-1, the proportion of plasmids in which DSGs were repaired was nearly the same under both permissive and restrictive conditions (5–10%). The results indicate that a disturbance in the function of the RAD55 gene, as in the RAD57 gene, leads to a drastic increase in the contribution of homology-dependent ligation to the repair of double-strand DNA breaks.     

Localized aggressive periodontitis is linked to human chromosome 1q25

Localized aggressive periodontitis (LAP; previously known as localized juvenile periodontitis) is one of the rapidly progressive periodontal diseases. Certain forms of familial LAP show a simple Mendelian pattern of transmission. However, no gene mutation has been identified to be responsible for the LAP phenotype. As an initial step to identify a gene mutation associated with LAP, we have performed genetic linkage analysis with four multigenerational families exhibiting the LAP phenotype. Affected individuals in the families were identified based on clinical and laboratory criteria in an attempt to define a homogeneous phenotype, since the clinical presentation of LAP may represent a manifestation of a heterogeneous group of diseases. The LAP phenotype is linked to a DNA marker, D1S492, with LOD score 3.48, =0.00. The haplotype analysis of the chromosome interval associated with D1S492 indicates that a LAP locus is located between D1S196 and D1S533 on chromosome 1, covering about 26 million DNA basepairs. We have also examined the DNA sequence of prostaglandin-endoperoxide synthase 2 (PTGS2 or cyclooxygenase 2, COX2) since prostaglandin 2 (PGE2), the product of COX2, is upregulated in LAP patients and COX2 is located between D1S196 and D1S533. No mutation in COX2 was identified in the patients.     

Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene

Summary A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per g DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus.     

Molecular cloning and characterization of the trpC gene from Penicillium chrysogenum

Summary We cloned the Penicillium chrysogenum trpC gene from a genomic library by complementation of an Escherichia coli trpC mutant lacking phosphoribosylanthranilate isomerase activity. The gene ecodes a 2.7 kb poly(A)⁺ RNA. We localized the gene by sequence analysis in a 2.9 kb DNA insert found in the smallest plasmid selected from the library. Sequence data strongly suggest that the organization of the gene is similar to that described in other Ascomycetes. We found that a DNA fragment which codes only for the carboxy-terminal protion of the polypeptide is sufficient for complementation of the E. coli trpC9830 mutation.     

A gene, SMP2, involved in plasmid maintenance and respiration in Saccharomyces cerevisiae encodes a highly charged protein

Summary The smp2 mutant of Saccharomyces cerevisiae shows increased stability of the heterologous plasmid pSR1 and YRp plasmids. A DNA fragment bearing the SMP2 gene was cloned by its ability to complement the slow growth of the smp2 smp3 double mutant (smp3 is another mutation conferring increased stability of plasmid pSR1). The nucleotide sequence of SMP2 indicated that it encodes a highly charged 95 kDa protein. Disruption of the genomic SMP2 gene resulted in a respiration-deficient phenotype, although the cells retained mitochondrial DNA, and showed increased stability of pSR1 like the original smp2 mutant. The fact that the smp2 mutant is not always respiration deficient and shows increased pSR1 stability even in a rho ⁰ strain lacking mitochondrial DNA suggested that the function of the Smp2 protein in plasmid maintenance is independent of respiration. The SMP2 locus was mapped at a site 71 cM from lys7 and 21 cM from ilv2/SMR1 on the right arm of chromosome XIII.     

Efficient DNA pairing in a Neurospora mutant defective in chromosome pairing

Summary A Neurospora crassa mutation, mei-2, affecting recombination and pairing of homologous chromosomes during meiosis, was characterized for its effect on repeat-induced point mutation (RIP). We found that RIP, which depends on recognition of DNA sequence homology, is not inhibited by mei-2, suggesting that the defect in chromosome pairing of this mutant is not due to a defect in DNA pairing and that DNA pairing is not dependent on chromosome pairing.     

Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points

Summary A pBR322-derived plasmid pKEN221 carrying a Serratia marcescens lpp gene overproduces the outer membrane lipoprotein in an Escherichia coli lpp ^–cell. However, when this strain was continuously cultured in a rich medium for about thirty generations, many Lpp^– mutants were accumulated. Out of six mutants analyzed, three were found to carry insertion mutation in the lpp gene in pKEN221. From restriction enzyme mapping and hybridization analysis of the mutant plasmid DNA, it was found that two mutants were caused by insertion sequence IS1 and one by IS5. Nucleotide sequence analysis of these mutant DNAs revealed that both IS1 and IS5 insertions occured in the A-T rich 5 untranslated region of the lpp gene. While the IS1 insertion resulted in a direct duplication of a nine-base-pair sequence in the original pKEN221 DNA at the junction with IS1, the IS5 insertion resulted in a direct duplication of a four-base-pair sequence. IS5 was found to contain inverted-repeat sequences of twelve nucleotides at its exact ends. This is the first example of the nucleotide sequence analysis of an IS5 insertion mutation. By Southern blot hybridization, the E. coli chromosomal DNA was found to contain about ten copies of IS5.