Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.     

Plasmid genes increase membrane permeability in Escherichia coli

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.     

Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital amaurosis

Leber congenital amaurosis (LCA) is one of the main causes of childhood blindness. To date, mutations in eight genes have been described, which together account for approximately 45% of LCA cases. We localized the genetic defect in a consanguineous LCA-affected family from Quebec and identified a splice defect in a gene encoding a centrosomal protein (CEP290). The defect is caused by an intronic mutation (c.2991+1655A-->G) that creates a strong splice-donor site and inserts a cryptic exon in the CEP290 messenger RNA. This mutation was detected in 16 (21%) of 76 unrelated patients with LCA, either homozygously or in combination with a second deleterious mutation on the other allele. CEP290 mutations therefore represent one of the most frequent causes of LCA identified so far.     

Viability of poliovirus/rhinovirus VPg chimeric viruses and identification of an amino acid residue in the VPg gene critical for viral RNA replication

Picornaviral RNA replication utilizes a small virus-encoded protein, termed 3B or VPg, as a primer to initiate RNA synthesis. This priming step requires uridylylation of the VPg peptide by the viral polymerase protein 3D(pol), in conjunction with other viral or host cofactors. In this study, we compared the viral specificity in 3D(pol)-catalyzed uridylylation reactions between poliovirus (PV) and human rhinovirus 16 (HRV16). It was found that HRV16 3D(pol) was able to uridylylate PV VPg as efficiently as its own VPg, but PV 3D(pol) could not uridylylate HRV16 VPg. Two chimeric viruses, PV containing HRV16 VPg (PV/R16-VPg) and HRV16 containing PV VPg (R16/PV-VPg), were constructed and tested for replication capability in H1-HeLa cells. Interestingly, only PV/R16-VPg chimeric RNA produced infectious virus particles upon transfection. No viral RNA replication or cytopathic effect was observed in cells transfected with R16/PV-VPg chimeric RNA, despite the ability of HRV16 3D(pol) to uridylylate PV VPg in vitro. Sequencing analysis of virion RNA isolated from the virus particles generated by PV/R16-VPg chimeric RNA identified a single residue mutation in the VPg peptide (Glu(6) to Val). Reverse genetics confirmed that this mutation was highly compensatory in enhancing replication of the chimeric viral RNA. PV/R16-VPg RNA carrying this mutation replicated with similar kinetics and magnitude to wild-type PV RNA. This cell culture-induced mutation in HRV16 VPg moderately increased its uridylylation by PV 3D(pol) in vitro, suggesting that it might be involved in other function(s) in addition to the direct uridylylation reaction. This study demonstrated the use of chimeric viruses to characterize viral specificity and compatibility in vivo between PV and HRV16 and to identify critical amino acid residue(s) for viral RNA replication.     

Recombination and oligonucleotide analysis of guanidine-resistant foot-and-mouth disease virus mutants.

Guanidine resistance (gr) mutations of foot-and-mouth disease virus were mapped by recombining pairs of temperature-sensitive mutants belonging to different subtypes. In each cross, one parent possessed a gr mutation. Recombinants were isolated by selection at the nonpermissive temperature and assayed for the ability to grow in the presence of guanidine. From the progeny of three crosses, four different types of recombinant were distinguished on the basis of protein composition and RNA fingerprint. The sequences of the RNase T1-resistant oligonucleotides were determined and located in the full-length sequence of foot-and-mouth disease virus. The resulting maps show that (i) each recombinant was generated by a single genetic crossover, and (ii) both of the gr mutations studied were located within an internal 2.9-kilobase region which spans the P34 gene. This supports our hypothesis that guanidine inhibits the growth of foot-and-mouth disease virus by acting on nonstructural polypeptide P34. Additional evidence was provided by RNA fingerprinting gr mutants. In two of four cases the gr mutation was associated with a change in an oligonucleotide located near the 3' end of the P34 gene; in one of these the nucleotide substitution was identified.     

A transposon insertion in the Arabidopsis SSR16 gene causes an embryo-defective lethal mutation

The SSR16 gene of Arabidopsis has been identified as a gene encoding a ribosomal protein S16 homolog through analysis of a transposon insertion mutation. The insertion mutation is lethal, arresting embryonic development at approximately the transition from the globular to the heart stage of embryonic development. Co-segregation of the mutant phenotype with the transposon-borne drug-resistance marker and loss of the inserted transposon concomitant with phenotypic reversion provided evidence that the transposon had caused the mutation. Sequences flanking the insertion site were amplified from DNA of viable heterozygotes by thermal asymmetric interlaced (TAIL) PCR. The amplified fragment flanking the 3' end of the inserted element was sequenced and found to be identical to an Arabidopsis expressed sequence tag (EST). The EST, in turn, contained a coding sequence homologous to the ribosomal protein S16 (RPS16) of bacteria such as Escherichia coli, Bacillus subtilis and Salmonella typhimurium , as well as Neurospora crassa mitochondria and higher plant plastids. Thus the gene identified by the embryo-defective lethal insertion mutation encodes an RPS16 homolog and has been designated the SSR16 gene.     

Mutants with base changes at the 3'-end of the 16S RNA from Escherichia coli. Construction, expression and functional analysis

The functionally important 3' domain of the ribosomal 16S RNA was altered by in vitro DNA manipulations of a plasmid-encoded 16S RNA gene. By in vitro DNA manipulations two double mutants were constructed in which C1399 was converted to A and G1401 was changed to either U or C and a single point mutant was made wherein G1416 was changed to U. Only one of the mutated rRNA genes could be cloned in a plasmid under the control of the natural rrnB promoters (U1416) whereas all three mutations were cloned in a plasmid under the control of the lambda PL promoter. In a strain coding for the temperature-sensitive lambda repressor cI857 the mutant RNAs could be expressed conditionally. We could show that all three mutant rRNAs were efficiently incorporated into 30S ribosomes. However, all three mutants inhibited the formation of stable 70S particles to various degrees. The amounts of mutated rRNAs were quantified by primer extension analysis which enabled us to assess the proportion of the mutated ribosomes which are actively engaged in in vivo protein biosynthesis. While ribosomes carrying the U1416 mutation in the 16S RNA were active in vivo a strong selection against ribosomes with the A1399/U1401 mutation in the 16S RNA from the polysome fraction is apparent. Ribosomes with 16S RNA bearing the A1399/C1401 mutation did not show a measurable protein biosynthesis activity in vivo. The growth rate of cells harbouring the different mutations reflected the in vivo translation capacities of the mutant ribosomes. The results underline the importance of the highly conserved nucleotides in the 3' domain of the 16S RNA for ribosomal function.     

Chemical and functional characterization of an altered form of ribosomal protein S4 derived from a strain of E. coli defective in auto-regulation of the alpha operon.

We have isolated a mutant form of Escherichia coli ribosomal protein S4. This mutant is temperature sensitive and apparently fails to autogenously regulate the gene products of the alpha operon, which consists of the genes for proteins S13, S11, S4, L17, and the alpha subunit of RNA polymerase (1). We have shown that this mutation results in the production of an S4 protein with a molecular weight approximately 4,000 daltons less than the wild-type protein. Our chemical analyses demonstrate that the mutant protein is missing its C-terminal section consisting of residues 170-203. However, our studies to determine the capacity of this mutant protein to bind 16S RNA show that this protein is unimpaired in RNA binding function. This observation suggests that the functional domain of protein S4 responsible for translational regulation of the S4 gene products requires more of the protein than the 16S RNA binding domain.     

Mitochondrial 12S rRNA gene mutations affect RNA secondary structure and lead to variable penetrance in hearing impairment

Mutations in the mitochondrial DNA are one of the most important causes of sensorineural hearing loss, especially in the 12S ribosomal RNA (rRNA) gene. We have analyzed the mtDNA 12S rRNA gene in a cohort of 443 families with hearing impairment, and have identified the A1555G mutation in 69 unrelated cases. A1555G is not a fully penetrant change, since only 63% of subjects with this change have developed hearing impairment. In addition, only 22% of the 183 A1555G deaf subjects were treated with aminoglycosides. Two novel nucleotide changes (T1291C and T1243C) were identified. T1243C was found in five deafness cases and one control sample. Mutation T1291C was detected in all maternally related individuals of a pedigree and in none of 95 control samples. Conservation analysis and comparison of the 12S rRNA structure with the 16S rRNA of Escherichia coli showed that the T at nucleotide 1243 and A at nucleotide 1555 are conserved positions. Prediction of RNA secondary structure showed changes in all 12S rRNA variants, the most severe being for T1291C. The reported data confirm the high prevalence of mutation A1555G in deafness cases and the major role of the 12S rRNA gene in hearing. The two novel changes reported here might have different contributions as deafness-related variants. T1291C fulfills the criteria of a disease-causing change. As in the case of mutation A1555G, the underlying phenotype of T1291C is not homogeneous for all family members, providing evidence for the implication of environmental and/or additional genetic factors.     

Myotone Dystrophien – und ihre Differenzialdiagnosen

The classic myotonic dystrophy, Steinert’s disease (DM1) was first described in 1909, and the second type, Ricker’s disease (DM2), in 1994. In 1992 the disease-causing mutation in DM1 was identified as a CTG repeat in the DMPK gene on chromosome 19q, and in 2001 the DM2 mutation was identified as a CCTG repeat expansion in the ZNF9 gene on chromosome 3q. Multisystemic symptoms of the diseases affect skeletal muscle, brain, eye, heart, and the endocrine system. The pathogenesis of both forms seems to be based on a gain-of-function RNA mechanism and on alterations in RNA metabolism and spliceopathy. Our review focuses on clinical features, diagnostic techniques, and new aspects of molecular pathogenesis and therapy.     

A new mtDNA-tRNA(Glu) mutation (14728T>C) presenting a late-onset mitochondrial encephalomyopathy

We identified a new mutation in the mtDNA-encoded transfer RNA glutamate gene (tRNAGlu) in a patient presenting with late-onset myopathy. The mutation was nearly homoplasmic in muscle but hardly detectable in peripheral blood. Adding to the list of disease-related mtDNA variants, our findings propose to consider screening of tRNAGlu in cases of late-onset neuromuscular disorders.     

Involvement of the Escherichia coli folate-binding protein YgfZ in RNA modification and regulation of chromosomal replication initiation

The Escherichia coli hda gene codes for a DnaA-related protein that is essential for the regulatory inactivation of DnaA (RIDA), a system that controls the initiation of chromosomal replication. We have identified the ygfZ gene, which encodes a folate-binding protein, as a suppressor of hda mutations. The ygfZ null mutation suppresses an hda null mutation. The over-initiation and abortive elongation phenotypes conferred by the hda mutations are partially suppressed in an hda ygfZ background. The accumulation of the active form of DnaA, ATP-DnaA, in the hda mutant is suppressed by the disruption of the ygfZ gene, indicating that YgfZ is involved in regulating the level of ATP-DnaA. Although ygfZ is not an essential gene, the ygfZ disruptant grows slowly, especially at low temperature, demonstrating that this gene is important for cellular proliferation. We have identified mnmE (trmE) as a suppressor of the ygfZ disruption. This gene encodes a GTPase involved in tRNA modification. Examination of RNA modification in the ygfZ mutant reveals reduced levels of 2-methylthio N(6)-isopentenyladenosine [corrected] indicating that YgfZ participates in the methylthio-group formation of this modified nucleoside in some tRNAs. These results suggest that YgfZ is a key factor in regulatory networks that act via tRNA modification.     

Codon choice and potential complementarity between mRNA downstream of the initiation codon and bases 1471-1480 in 16S ribosomal RNA affects expression of glnS.

A cis-acting expression mutation, GAG to GAA, in the third codon of the glnS gene is analyzed. Both codons code for glutamic acid but the mutation is known to increase gene expression by four fold. We show that the mutation has an effect only if it is located in the beginning of a gene but not if located internally. Data are presented that suggest that the reason for the increased expression by the mutation is the potential formation of one more base pair between the mRNA and 16S ribosomal RNA. Gene expression varies about 16 fold as the number of potential base pairs within the sequence 1471-1480 in 16S RNA increase from two to ten. We also give evidence that supports the idea that the presence of rare codons near the beginning of the mRNA can affect expression.