MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection

MmeI is an unusual Type II restriction enzyme that is useful for generating long sequence tags. We have cloned the MmeI restriction-modification (R-M) system and found it to consist of a single protein having both endonuclease and DNA methyltransferase activities. The protein comprises an amino-terminal endonuclease domain, a central DNA methyltransferase domain and C-terminal DNA recognition domain. The endonuclease cuts the two DNA strands at one site simultaneously, with enzyme bound at two sites interacting to accomplish scission. Cleavage occurs more rapidly than methyl transfer on unmodified DNA. MmeI modifies only the adenine in the top strand, 5′-TCCRAC-3′. MmeI endonuclease activity is blocked by this top strand adenine methylation and is unaffected by methylation of the adenine in the complementary strand, 5′-GTYGGA-3′. There is no additional DNA modification associated with the MmeI R-M system, as is required for previously characterized Type IIG R-M systems. The MmeI R-M system thus uses modification on only one of the two DNA strands for host protection. The MmeI architecture represents a minimal approach to assembling a restriction-modification system wherein a single DNA recognition domain targets both the endonuclease and DNA methyltransferase activities

Transglutaminase 6: a protein associated with central nervous system development and motor function.

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second β-barrel domain in man and a variant with truncated β-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca(2+) and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca(2+) and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons

Two isoforms of tissue transglutaminase mediate opposing cellular fates

Opposing cellular responses are typically regulated by distinct sets of genes. However, tissue transglutaminase (TGase) provides an interesting example of a single gene product that has been implicated both in affording protection against cellular insults as well as in promoting cell death. Here, we shed some light on how these conflicting activities might be manifested by demonstrating that alternative transcripts of TGase differentially affect cell viability. We show that although the full-length TGase protein affords strong protection against cell death signals, a shorter version of TGase that is truncated at the 3′ end, and thus called TGase-short (TGase-S), is cytotoxic. The apoptotic activity of TGase-S is not dependent on its transamidation activity because the mutation of a cysteine residue that is essential for catalyzing this reaction does not compromise the ability of TGase-S to induce cell death. Intriguingly, TGase-S undergoes inappropriate oligomer formation in cells before cell death, suggesting a novel mechanism for the apoptotic effects of this protein

Increased trabecular bone and improved biomechanics in an osteocalcin-null rat model created by CRISPR/Cas9 technology

Osteocalcin, also known as bone γ-carboxyglutamate protein (Bglap), is expressed by osteoblasts and is commonly used as a clinical marker of bone turnover. A mouse model of osteocalcin deficiency has implicated osteocalcin as a mediator of changes to the skeleton, endocrine system, reproductive organs and central nervous system. However, differences between mouse and human osteocalcin at both the genome and protein levels have challenged the validity of extrapolating findings from the osteocalcin-deficient mouse model to human disease. The rat osteocalcin (Bglap) gene locus shares greater synteny with that of humans. To further examine the role of osteocalcin in disease, we created a rat model with complete loss of osteocalcin using the CRISPR/Cas9 system. Rat osteocalcin was modified by injection of CRISPR/Cas9 mRNA into the pronuclei of fertilized single cell Sprague-Dawley embryos, and animals were bred to homozygosity and compound heterozygosity for the mutant alleles. Dual-energy X-ray absorptiometry (DXA), glucose tolerance testing (GTT), insulin tolerance testing (ITT), microcomputed tomography (µCT), and a three-point break biomechanical assay were performed on the excised femurs at 5 months of age. Complete loss of osteocalcin resulted in bones with significantly increased trabecular thickness, density and volume. Cortical bone volume and density were not increased in null animals. The bones had improved functional quality as evidenced by an increase in failure load during the biomechanical stress assay. Differences in glucose homeostasis were observed between groups, but there were no differences in body weight or composition. This rat model of complete loss of osteocalcin provides a platform for further understanding the role of osteocalcin in disease, and it is a novel model of increased bone formation with potential utility in osteoporosis and osteoarthritis research

Nuclear Structure Study of 104^{104}Pd by Coulomb Excitation at the Warsaw Heavy Ion Laboratory

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