Catherine M. Abbott

Mutations in Sox18 underlie cardiovascular and hair follicle defects in ragged mice.

Analysis of classical mouse mutations has been useful in the identification and study of many genes. We previously mapped Sox18, encoding an SRY-related transcription factor, to distal mouse chromosome 2 (ref. 2). This region contains a known mouse mutation, ragged (Ra), that affects the coat and vasculature. Here we have directly evaluated Sox18 as a candidate for Ra. We found that Sox18 is expressed in the developing vascular endothelium and hair follicles in mouse embryos. Furthermore, we found no recombination between Sox18 and Ra in an interspecific backcross segregating for the Ra phenotype. We found point mutations in Sox18 in two different Ra alleles that result in missense translation and premature truncation of the encoded protein. Fusion proteins containing these mutations lack the ability to activate transcription relative to wild-type controls in an in vitro assay. Our observations implicate mutations in Sox18 as the underlying cause of the Ra phenotype, and identify Sox18 as a critical gene for cardiovascular and hair follicle formation.

Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours

BackgroundThe tissue-specific translation elongation factor eEF1A2 was recently shown to be a potential oncogene that is overexpressed in ovarian cancer. Although there is no direct evidence for an involvement of eEF1A2 in breast cancer, the genomic region to which EEF1A2 maps, 20q13, is frequently amplified in breast tumours. We therefore sought to establish whether eEF1A2 expression might be upregulated in breast cancer.MethodseEF1A2 is highly similar (98%) to the near-ubiquitously expressed eEF1A1 (formerly known as EF1-α) making analysis with commercial antibodies difficult. We have developed specific anti-eEF1A2 antibodies and used them in immunohistochemical analyses of tumour samples. We report the novel finding that although eEF1A2 is barely detectable in normal breast it is moderately to strongly expressed in two-thirds of breast tumours. This overexpression is strongly associated with estrogen receptor positivity.ConclusioneEF1A2 should be considered as a putative oncogene in breast cancer that may be a useful diagnostic marker and therapeutic target for a high proportion of breast tumours. The oncogenicity of eEF1A2 may be related to its role in protein synthesis or to its potential non-canonical functions in cytoskeletal remodelling or apoptosis.

Genomic structure and chromosomal mapping of the murine and human Mbd1, Mbd2, Mbd3, and Mbd4 genes

Abstract. DNA methylation is essential for murine development and is implicated in the control of gene expression. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of mammalian, nuclear proteins related by the presence in each of an amino acid motif called the methyl-CpG binding domain (MBD). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA. MeCP2, MBD1 and MBD2 can also repress transcription. We describe the genomic structure and chromosomal localization of the human and murine Mbd1, Mbd2, Mbd3, and Mbd4 genes. We find that the highly similar MBD2 and MBD3 proteins are encoded by genes that map to different chromosomes in humans and mice but show a similar genomic structure. The Mbd1 and Mbd2 genes, in contrast, map together to murine and human Chromosomes (Chrs)18. The Mbd3 and Mbd4 genes map to murine Chrs 10 and 6, respectively, while the human MBD3 and MBD4 genes map to Chrs 19 and 3, respectively.

Mouse chromosome 2

mKimmel Cancer Center, Jefferson Medical College, Department of Microbiology and Immunology, 233 South 10th Street, Philadelphia, Pennsylvania 19107-5541, USA 2Human Genetics Unit, Molecular Medicine Center, The University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK 3The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA 4Departrnent of Animal Science, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0908, USA SMRC Mammalian Genetics Unit, Harwell, Didcot Oxon OX 11 ORD, UK

Mouse chromosome 4.

This year’s report incorporates 78 new genetic markers into the consensus linkage map. Of these markers, ten have a known, mapped human homolog. The murine gene, followed by the human homolog and the human chromosomal location in parenthesis are as follows: Cpt2 4 CPT2 (1p32); Cyp2j5 and Cyp2j6 4 CYP2J2 (1p31.3-p31.2); Guca1b 4 GUCA2B (1p34-p33); Htr6 4 HTR6 (1p36-1p35); Hub 4 ELAVL2 (9p21); Hud 4 ELAVL4 (1p34); Matn1 4 MATN1 (1p35); Mmp16 4 MMP16 (8q21.3-q22.1); Tgfbr1 4 TGFBR1 (9q33-q34). In the case of the Cyp genes, two genes have been identified in mouse while only a single gene has been identified in humans. Mouse Chromosome (Chr) 4 shares significant stretches of linkage homology with human Chr 1, 6, 8, 9 and 21 (34245; 23572). The entire distal half of mouse Chr 4 is homologous with human Chr 1p. There have been four changes in nomenclature of genetic loci on Chr 4. Gene names and/or symbols that have been changed by the Nomenclature Committee include: Cerr1 changed to Cer1; Dana changed to D4H1s1733E; Zie changed to Gklf; and Etl2 changed to Il11ra2.

The gene for human complement component C9 mapped to chromosome 5 by polymerase chain reaction

The gene for human complement component C9 has been mapped to chromosome 5. This was achieved by using a novel application of the polymerase chain reaction to amplify specifically the human C9 gene on a background of rodent DNA in somatic cell hybrids. The assignment to chromosome 5 was confirmed by in situ hybridization to human metaphase chromosomes, giving a regional localization of 5p13.

Structural models of human eEF1A1 and eEF1A2 reveal two distinct surface clusters of sequence variation and potential differences in phosphorylation.

Background Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1) and elongation factor 1 alpha-2 (eEF1A2) have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome. Methodology/Principal Findings To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D) models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Bα and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins. Conclusions The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these proteins. The predicted variant-specific phosphorylation sites also provide a basis for experimental verification by mutagenesis. The models provide a structural framework for interpretation of the resulting functional analysis.

Translation elongation factor eEF1A2 is essential for post-weaning survival in mice

Translation elongation factor eEF1A, formerly known as EF-1α, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.

The translation elongation factor eEF1A1 couples transcription to translation during heat shock response

Translation elongation factor eEF1A has a well-defined role in protein synthesis. In this study, we demonstrate a new role for eEF1A: it participates in the entire process of the heat shock response (HSR) in mammalian cells from transcription through translation. Upon stress, isoform 1 of eEF1A rapidly activates transcription of HSP70 by recruiting the master regulator HSF1 to its promoter. eEF1A1 then associates with elongating RNA polymerase II and the 3′UTR of HSP70 mRNA, stabilizing it and facilitating its transport from the nucleus to active ribosomes. eEF1A1-depleted cells exhibit severely impaired HSR and compromised thermotolerance. In contrast, tissue-specific isoform 2 of eEF1A does not support HSR. By adjusting transcriptional yield to translational needs, eEF1A1 renders HSR rapid, robust, and highly selective; thus, representing an attractive therapeutic target for numerous conditions associated with disrupted protein homeostasis, ranging from neurodegeneration to cancer. DOI: http://dx.doi.org/10.7554/eLife.03164.001

Two opposing effects of mismatch repair on CTG repeat instability in Escherichia coli

The expansion of normally polymorphic CTG microsatellites in certain human genes has been identified as the causative mutation of a number of hereditary neurological disorders, including Huntingtons disease and myotonic dystrophy. Here, we have investigated the effect of methyl‐directed mismatch repair (MMR) on the stability of a (CTG)43 repeat in Escherichia coli over 140 generations and find two opposing effects. In contrast to orientation‐dependent repeat instability in wild‐type E. coli and yeast, we observed no orientation dependence in MMR−E. coli cells and suggest that, for the repeat that we have studied, orientation dependence in wild‐type cells is mainly caused by functional mismatch repair genes. Our results imply that slipped structures are generated during replication, causing single triplet expansions and contractions in MMR− cells, because they are left unrepaired. On the other hand, we find that the repair of such slipped structures by the MMR system can go awry, resulting in large contractions. We show that these mutS‐dependent contractions arise preferentially when the CTG sequence is encoded by the lagging strand. The nature of this orientation dependence argues that the small slipped structures that are recognized by the MMR system are formed primarily on the lagging strand of the replication fork. It also suggests that, in the presence of functional MMR, removal of 3 bp slipped structures causes the formation of larger contractions that are probably the result of secondary structure formation by the CTG sequence. We rationalize the opposing effects of MMR on repeat tract stability with a model that accounts for CTG repeat instability and loss of orientation dependence in MMR− cells. Our work resolves a contradiction between opposing claims in the literature of both stabilizing and destabilizing effects of MMR on CTG repeat instability in E. coli.