Francisco Castillo

Prevalence and Evolutionary Origins of the del(GJB6-D13S1830) Mutation in the DFNB1 Locus in Hearing-Impaired Subjects: A Multicenter Study

Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%-50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in approximately 50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%-20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.

Mutations in the gene encoding pejvakin, a newly identified protein of the afferent auditory pathway, cause DFNB59 auditory neuropathy

Auditory neuropathy is a particular type of hearing impairment in which neural transmission of the auditory signal is impaired, while cochlear outer hair cells remain functional. Here we report on DFNB59, a newly identified gene on chromosome 2q31.1–q31.3 mutated in four families segregating autosomal recessive auditory neuropathy. DFNB59 encodes pejvakin, a 352-residue protein. Pejvakin is a paralog of DFNA5, a protein of unknown function also involved in deafness. By immunohistofluorescence, pejvakin is detected in the cell bodies of neurons of the afferent auditory pathway. Furthermore, Dfnb59 knock-in mice, homozygous for the R183W variant identified in one DFNB59 family, show abnormal auditory brainstem responses indicative of neuronal dysfunction along the auditory pathway. Unlike previously described sensorineural deafness genes, all of which underlie cochlear cell pathologies, DFNB59 is the first human gene implicated in nonsyndromic deafness due to a neuronal defect.

The Escherichia coli K‐12 sheA gene encodes a 34‐kDa secreted haemolysin

Haemolytic toxins belong to one of several classes of virulence factors that contribute to bacterial pathogenicity. The non‐pathogenic Escherichia coli K‐12 laboratory strain was considered for years to be non‐haemolytic. However, overproduction of several transcriptional regulators induced the appearance of a haemolytic activity that is absent under usual laboratory growth conditions. In this work, we have cloned and characterized an E. coli K‐12 gene, sheA, whose overexpression results in a haemolytic phenotype. It maps to min 27 on the genetic map, and codes for a 34‐kDa polypeptide with at least one putative transmembrane segment. This polypeptide, which has neither signal peptide nor other known secretory motifs, is secreted to the medium during the exponential growth phase. In vitro coupled transcription/translation assays, using a plasmid carrying only the sheA gene as template, resulted in the production of a polypeptide with haemolytic activity per se. Our results demonstrate that the sheA gene actually encodes the E. coli K‐12 chromosomal haemolysin. The SheA haemolysin does not resemble other known cytolytic toxins, and it may represent the prototype of a novel family, as suggested by the presence of homologues in several E. coli pathogenic strains and in Shigella flexneri.

Construction and Characterization of Mutations at Codon 751 of the Escherichia coli gyrB Gene That Confer Resistance to the Antimicrobial Peptide Microcin B17 and Alter the Activity of DNA Gyrase

Microcin B17 is a peptide antibiotic that inhibits DNA replication in Escherichia coli by targeting DNA gyrase. Previously, two independently isolated microcin B17-resistant mutants were shown to harbor the same gyrB point mutation that results in the replacement of tryptophan 751 by arginine in the GyrB polypeptide. We used site-directed mutagenesis to construct mutants in which tryptophan 751 was deleted or replaced by other amino acids. These mutants exhibit altered DNA gyrase activity and different levels of resistance to microcin B17.

Hearing Is Normal without Connexin30

Gjb2 and Gjb6, two contiguous genes respectively encoding the gap junction protein connexin26 (Cx26) and connexin 30 (Cx30) display overlapping expression in the inner ear. Both have been linked to the most frequent monogenic hearing impairment, the recessive isolated deafness DFNB1. Although there is robust evidence for the direct involvement of Cx26 in cochlear functions, the contribution of Cx30 is unclear since deletion of Cx30 strongly downregulates Cx26 both in human and in mouse. Thus, it is imperative that any role of Cx30 in audition be clearly evaluated. Here, we developed a new Cx30 knock-out mouse model (Cx30Δ/Δ) in which half of Cx26 expression was preserved. Our results show that Cx30 and Cx26 coordinated expression is dependent on the spacing of their surrounding chromosomic region, and that Cx30Δ/Δ mutants display normal hearing. Thus, in deaf patients with GJB6 deletion as well as in the previous Cx30 knock-out mouse model, defective Cx26 expression is the likely cause of deafness, and in contrast to current opinion, Cx30 is dispensable for cochlear functions.

Consortin, a trans-Golgi network cargo receptor for the plasma membrane targeting and recycling of connexins

Targeting of numerous transmembrane proteins to the cell surface is thought to depend on their recognition by cargo receptors that interact with the adaptor machinery for anterograde traffic at the distal end of the Golgi complex. We report here on consortin, a novel integral membrane protein that is predicted to be intrinsically disordered, i.e. that contains large segments whose native state is unstructured. We identified consortin as a binding partner of connexins, the building blocks of gap junctions. Consortin is located at the trans-Golgi network (TGN), in tubulovesicular transport organelles, and at the plasma membrane. It directly interacts with the TGN clathrin adaptors GGA1 and GGA2, and disruption of this interaction by expression of a consortin mutant lacking the acidic cluster-dileucine (DXXLL) GGA interaction motif causes an intracellular accumulation of several connexins. RNA interference-mediated silencing of consortin expression in HeLa cells blocks the cell surface targeting of these connexins, which accumulate intracellularly, whereas partial depletion and redistribution of the consortin pool slows down the intracellular degradation of gap junction plaques. Altogether, our results show that, by studying connexin trafficking, we have identified the first TGN cargo receptor for the targeting of transmembrane proteins to the plasma membrane. The identification of consortin provides in addition a potential target for therapies aimed at diseases in which connexin traffic is altered, including cardiac ischemia, peripheral neuropathies, cataracts and hearing impairment. Sequence accession numbers. GenBank: Human CNST cDNA, NM_152609; mouse Cnst cDNA, NM_146105.

Characterization of the Pseudomonas pseudoalcaligenes CECT5344 Cyanase, an Enzyme That Is Not Essential for Cyanide Assimilation

ABSTRACT Cyanase catalyzes the decomposition of cyanate into CO2 and ammonium, with carbamate as an unstable intermediate. The cyanase of Pseudomonas pseudoalcaligenes CECT5344 was negatively regulated by ammonium and positively regulated by cyanate, cyanide, and some cyanometallic complexes. Cyanase activity was not detected in cell extracts from cells grown with ammonium, even in the presence of cyanate. Nevertheless, a low level of cyanase activity was detected in nitrogen-starved cells. The cyn gene cluster of P. pseudoalcaligenes CECT5344 was cloned and analyzed. The cynA, cynB, and cynD genes encode an ABC-type transporter, the cynS gene codes for the cyanase, and the cynF gene encodes a novel σ54-dependent transcriptional regulator which is not present in other bacterial cyn gene clusters. The CynS protein was expressed in Escherichia coli and purified by following a simple and rapid protocol. The P. pseudoalcaligenes cyanase showed an optimal pH of 8.5°C and a temperature of 65°C. An insertion mutation was generated in the cynS gene. The resulting mutant was unable to use cyanate as the sole nitrogen source but showed the same resistance to cyanate as the wild-type strain. These results, in conjunction with the induction pattern of the enzymatic activity, suggest that the enzyme has an assimilatory function. Although the induction of cyanase activity in cyanide-degrading cells suggests that some cyanate may be generated from cyanide, the cynS mutant was not affected in its ability to degrade cyanide, which unambiguously indicates that cyanate is not a central metabolite in cyanide assimilation.

Structure of p15 PAF –PCNA complex and implications for clamp sliding during DNA replication and repair

The intrinsically disordered protein p15(PAF) regulates DNA replication and repair by binding to the proliferating cell nuclear antigen (PCNA) sliding clamp. We present the structure of the human p15(PAF)-PCNA complex. Crystallography and NMR show the central PCNA-interacting protein motif (PIP-box) of p15(PAF) tightly bound to the front-face of PCNA. In contrast to other PCNA-interacting proteins, p15(PAF) also contacts the inside of, and passes through, the PCNA ring. The disordered p15(PAF) termini emerge at opposite faces of the ring, but remain protected from 20S proteasomal degradation. Both free and PCNA-bound p15(PAF) binds DNA mainly through its histone-like N-terminal tail, while PCNA does not, and a model of the ternary complex with DNA inside the PCNA ring is consistent with electron micrographs. We propose that p15(PAF) acts as a flexible drag that regulates PCNA sliding along the DNA and facilitates the switch from replicative to translesion synthesis polymerase binding.

Regulation and Characterization of Two Nitroreductase Genes, nprA and nprB, of Rhodobacter capsulatus

ABSTRACT Among photosynthetic bacteria, strains B10 and E1F1 of Rhodobacter capsulatus photoreduce 2,4-dinitrophenol (DNP), which is stoichiometrically converted into 2-amino-4-nitrophenol by a nitroreductase activity. The reduction of DNP is inhibited in vivo by ammonium, which probably acts at the level of the DNP transport system and/or physiological electron transport to the nitroreductase, since this enzyme is not inhibited by ammonium in vitro. Using the complete genome sequence data for strain SB1003 of R. capsulatus, two putative genes coding for possible nitroreductases were isolated from R. capsulatus B10 and disrupted. The phenotypes of these mutant strains revealed that both genes are involved in the reduction of DNP and code for two major nitroreductases, NprA and NprB. Both enzymes use NAD(P)H as the main physiological electron donor. The nitroreductase NprA is under ammonium control, whereas the nitroreductase NprB is not. In addition, the expression of the nprB gene seems to be constitutive, whereas nprA gene expression is inducible by a wide range of nitroaromatic and heterocyclic compounds, including several dinitroaromatics, nitrofuran derivatives, CB1954, 2-aminofluorene, benzo[a]pyrene, salicylic acid, and paraquat. The identification of two putative mar/sox boxes in the possible promoter region of the nprA gene and the induction of nprA gene expression by salicylic acid and 2,4-dinitrophenol suggest a role in the control of the nprA gene for the two-component MarRA regulatory system, which in Escherichia coli controls the response to some antibiotics and environmental contaminants. In addition, upregulation of the nprA gene by paraquat indicates that this gene is probably a member of the SoxRS regulon, which is involved in the response to stress conditions in other bacteria.

INHIBITION OF ACONITASE AND FUMARASE BY NITROGEN COMPOUNDS IN RHODOBACTER CAPSULATUS

High levels of aconitase and fumarase activities were found in Rhodobacter capsulatus E1F1 cells cultured with nitrate as the sole nitrogen source either under light-anaerobic or dark-aerobic conditions. Both activities were strongly and reversibly inhibited in vitro by nitrite or nitric oxide, whereas nitrate or hydroxylamine showed a lower effect. Other enzymes of the tricarboxylic acids cycle such as malate dehydrogenase or isocitrate dehydrogenase were not affected by these nitrogen compounds. When growing on nitrate in the dark R. capsulatus E1F1 cells accumulated nitrite intracellularly, so that an in vivo inhibition of aconitase and fumarase could account for the strong inhibition of growth observed in the presence of nitrite under dark-aerobic conditions.