Intracellular Transport, Assembly, and Degradation of Wild-Type and Disease-linked Mutant Gap Junction Proteins

More than 130 different mutations in the gap junction integral plasma membrane protein connexin32 (Cx32) have been linked to the human peripheral neuropathy X-linked Charcot–Marie–Tooth disease (CMTX). How these various mutants are processed by the cell and the mechanism(s) by which they cause CMTX are unknown. To address these issues, we have studied the intracellular transport, assembly, and degradation of three CMTX-linked Cx32 mutants stably expressed in PC12 cells. Each mutant had a distinct fate: E208K Cx32 appeared to be retained in the endoplasmic reticulum (ER), whereas both the E186K and R142W mutants were transported to perinuclear compartments from which they trafficked either to lysosomes (R142W Cx32) or back to the ER (E186K Cx32). Despite these differences, each mutant was soluble in nonionic detergent but unable to assemble into homomeric connexons. Degradation of both mutant and wild-type connexins was rapid (t1/2 \u3c 3 h) and took place at least in part in the ER by a process sensitive to proteasome inhibitors. The mutants studied are therefore unlikely to cause disease by accumulating in degradation-resistant aggregates but instead are efficiently cleared from the cell by quality control processes that prevent abnormal connexin molecules from traversing the secretory pathway

Mutagenicity of Acridines in a Reversion Assay Based on Tetracycline Resistance in Plasmid pBR322 in Escherichia Coli

The mutagenicity of a series of acridine compounds was studied in an assay based on the reversion of mutations in the tetracycline-resistance gene (tet) of plasmid pBR322 in Escherichia coli. Mutations that restore the tetracycline-resistant phenotype were detected in tetracycline-sensitive strains carrying mutant plasmids. Mutations that revert by +2, +1, −1, and −2 frameshift mutations and by base-pair substitutions were used to analyze the mutagenicity of two simple acridines, two acridine mustards, and a nitroacridine. The simple acridines (9-aminoacridine and quinacrine) effectively induced −1 frameshifts and weakly induced +1 frameshifts. The acridine mustards (quinacrine mustard and ICR-191) were more potent inducers of −1 and +1 frameshifts than the simple acridines. Reactive acridines, including both the mustards and the nitroacridine Entozon, were effective inducers of −2 frameshifts but the simple acridines were not. The two classes of reactive acridines differed from one another, in that the mustards were better inducers of +1 frameshifts than Entozon, whereas Entozon was a particularly potent inducer of −2 frameshifts. None of the compounds induced +2 frameshifts, and the induction of base-pair substitutions was negligible. These results confirm and extend studies showing that adduct-forming acridines are stronger frameshift mutagens than simple intercalating acridines and that the acridines differ from one another not only in overall mutagenic potency but also in the prevalence of different classes of frameshift mutations

Connexin32 and X-Linked Charcot-Marie-Tooth Disease

This paper deals with the genetic defect responsible for the X-linked form of Charcot-Marie-Tooth disease.Mutations in the gap junction geneconnexin32(Cx32) cause the X-linked form of Charcot–Marie–Tooth disease, an inherited demyelinating neuropathy

Altered Trafficking of Mutant Connexin32

We examined the cellular localization of nine different connexin32 (Cx32) mutants associated with X-linked Charcot–Marie–Tooth disease (CMTX) in communication-incompetent mammalian cells. Cx32 mRNA was made, but little or no protein was detected in one class of mutants. In another class of mutants, Cx32 protein was detectable in the cytoplasm and at the cell surface, where it appeared as plaques and punctate staining. Cx32 immunoreactivity in a third class of mutants was restricted to the cytoplasm, where it often colocalized with the Golgi apparatus. Our studies suggest that CMTX mutations have a predominant effect on the trafficking of Cx32 protein, resulting in a potentially toxic cytoplasmic accumulation of Cx32 in these cells. These results and evidence of cytoplasmic accumulation of other mutated myelin proteins suggest that diseases affecting myelinating cells may share a common pathophysiology

Connexin32 is a Myelin-Related Protein in the PNS and CNS

We have examined the expression of a gap junction protein, connexin32 (Cx32), in Schwann cells and oligodendrocytes. In peripheral nerve, Cx32 is found in the paranodal myelin loops and Schmidt-Lanterman incisures of myelinating Schwann cells, and the levels of Cx32 protein and mRNA change in parallel with those of other myelin-related genes during development, Wallerian degeneration, and axonal regeneration. In the central nervous system, Cx32 is found in oligodendrocytes and their processes, but not in compact myelin, and the levels of Cx32 protein and mRNA increase during development in parallel with those of the other myelin genes. Thus, Cx32 is expressed as part of the myelinating phenotype of both Schwann cells and oligodendrocytes, indicating that this gap junction protein plays in important role in the biology of myelin-forming cells

The E705K Mutation in hPMS2 Exerts Recessive, Not Dominant, Effects on Mismatch Repair

The hPMS2 mutation E705K is associated with Turcot syndrome. To elucidate the pathogenesis of hPMS2-E705K, we modeled this mutation in yeast and characterized its expression and effects on mutation avoidance in mammalian cells. We found that while hPMS2-E705K (pms1-E738K in yeast) did not significantly affect hPMS2 (Pms1p in yeast) stability or interaction with MLH1, it could not complement the mutator phenotype in MMR-deficient mouse or yeast cells. Furthermore, hPMS2-E705K/pms1-E738K inhibited MMR in wild-type (WT) mammalian cell extracts or yeast cells only when present in excess amounts relative to WT PMS2. Our results strongly suggest that hPMS2-E705K is a recessive loss-of-function allele

A measurement of the millimetre emission and the Sunyaev-Zel'dovich effect associated with low-frequency radio sources

We present a statistical analysis of the millimetre-wavelength properties of 1.4GHz-selected sources and a detection of the Sunyaev–Zel’dovich (SZ) effect associated with the haloes that host them. We stack data at 148, 218 and 277GHz from the Atacama Cosmology Telescope at the positions of a large sample of radio AGN selected at 1.4GHz. The thermal SZ effect associated with the haloes that host the AGN is detected at the 5σ level through its spectral signature, representing a statistical detection of the SZ effect in some of the lowest mass haloes (average M 200 ≈ 10 13 M. h −1 70 ) studied to date. The relation between the SZ effect and mass (based on weak lensing measurements of radio galaxies) is consistent with that measured by Planck for local bright galaxies. In the context of galaxy evolution models, this study confirms that galaxies with radio AGN also typically support hot gaseous haloes. Adding Herschel observations allows us to show that the SZ signal is not significantly contaminated by dust emission. Finally, we analyse the contribution of radio sources to the angular power spectrum of the cosmic microwave background

Mutations Affecting a Putative MutLa Endonuclease Motif Impact Multiple DNA Mismatch Repair Functions

Mutations in DNA mismatch repair (MMR) lead to increased mutation rates and higher recombination between similar, but not identical sequences, as well as resistance to certain DNA methylating agents. Recently, a component of human MMR machinery, MutLα, has been shown to display a latent endonuclease activity. The endonuclease active site appears to include a conserved motif, DQHA(X)2E(X)4E, within the COOH-terminus of human PMS2. Substitution of the glutamic acid residue (E705) abolished the endonuclease activity and mismatch-dependent excision in vitro. Previously, we showed that the PMS2-E705K mutation and the corresponding mutation in Saccharomyces cerevisiae were both recessive loss of function alleles for mutation avoidance in vivo. Here, we show that mutations impacting this endonuclease motif also significantly affect MMR-dependent suppression of homeologous recombination in yeast and responses to Sn1-type methylating agents in both yeast and mammalian cells. Thus, our in vivo results suggest that the endonuclease activity of MutLα is important not only in MMR-dependent mutation avoidance but also for recombination and damage response functions

Which Came First, the Mutation or the Antibiotic?: Understanding Antibiotic Resistance Through Fluctuation Analysis

This case study presents the story of Phil, an undergraduate majoring in biology, whose Russian cousin Dimitri has contracted tuberculosis (TB) from inmates at the prison where he works. Phil learns that his cousin\u27s failure to complete his antibiotic regimen likely contributed to the evolution of antibiotic-resistant TB in his body. Phil consults with his friend Stacy, and together they try to understand Dimitri\u27s condition by applying what they are learning in their genetics lab experiment about the role of random mutation in bacterial evolution (including the development of antibiotic resistance) through Luria- Delbrück fluctuation analysis. The same analysis includes calculation of the mutation rate, which Phil realizes is sufficient to cause MRSA and other antibiotic-resistant infections. This case study was originally developed for concurrent use in freshman/sophomore-level genetics, elementary statistics, and precalculus. However, it is also very appropriate for courses in introductory biology, evolutionary biology, and biostatistics. The teaching notes discuss various ways to run the case depending on the mathematics and biology background of students

Mammalian DNA Mismatch Repair

Details the process of DNA mismatch repair in mammals (MMR). Mismatch repair in Escherichia coli and in eukaryotes; Phenotypes of MMR gene defiencies; Role of MMR genes in mouse tumorigenesis; Role of MMR genes in mutation avoidance; Other phenotypes associated with MMR gene defects; Defining the relationship between the functions of MMR in mutation and tumor avoidance