Lois Balmer

The influence of ontogeny and light environment on the expression of visual pigment opsins in the retina of the black bream, Acanthopagrus butcheri

SUMMARY The correlation between ontogenetic changes in the spectral absorption characteristics of retinal photoreceptors and expression of visual pigment opsins was investigated in the black bream, Acanthopagrus butcheri. To establish whether the spectral qualities of environmental light affected the complement of visual pigments during ontogeny, comparisons were made between fishes reared in: (1) broad spectrum aquarium conditions; (2) short wavelength-reduced conditions similar to the natural environment; or (3) the natural environment (wild-caught). Microspectrophotometry was used to determine the wavelengths of spectral sensitivity of the photoreceptors at four developmental stages: larval, post-settlement, juvenile and adult. The molecular sequences of the rod (Rh1) and six cone (SWS1, SWS2A and B, Rh2Aα and β, and LWS) opsins were obtained and their expression levels in larval and adult stages examined using quantitative RT-PCR. The changes in spectral sensitivity of the cones were related to the differing levels of opsin expression during ontogeny. During the larval stage the predominantly expressed opsin classes were SWS1, SWS2B and Rh2Aα, contrasting with SWS2A, Rh2Aβ and LWS in the adult. An increased proportion of long wavelength-sensitive double cones was found in fishes reared in the short wavelength-reduced conditions and in wild-caught animals, indicating that the expression of cone opsin genes is also regulated by environmental light.

Identification of a Novel AU-Rich Element in the 3′ Untranslated Region of Epidermal Growth Factor Receptor mRNA That Is the Target for Regulated RNA-Binding Proteins

ABSTRACT The epidermal growth factor receptor (EGF-R) plays an important role in the growth and progression of estrogen receptor-negative human breast cancers. EGF binds with high affinity to the EGF-R and activates a variety of second messenger pathways that affect cellular proliferation. However, the underlying mechanisms involved in the regulation of EGF-R expression in breast cancer cells are yet to be described. Here we show that the EGF-induced upregulation of EGF-R mRNA in two human breast cancer cell lines that overexpress EGF-R (MDA-MB-468 and BT-20) is accompanied by stabilization (>2-fold) of EGF-R mRNA. Transient transfections using a luciferase reporter identified a novel EGF-regulated ∼260-nucleotide (nt)cis-acting element in the 3′ untranslated region (3′-UTR) of EGF-R mRNA. This cis element contains two distinct AU-rich sequences (∼75 nt), EGF-R1A with two AUUUA pentamers and EGF-R2A with two AUUUUUA extended pentamers. Each independently regulated the mRNA stability of the heterologous reporter. Analysis of mutants of the EGF-R2A AU-rich sequence demonstrated a role for the 3′ extended pentamer in regulating basal turnover. RNA gel shift analysis identified cytoplasmic proteins (∼55 to 80 kDa) from breast cancer cells that bound specifically to the EGF-R1A and EGF-R2A cis-acting elements and whose binding activity was rapidly downregulated by EGF and phorbol esters. RNA gel shift analysis of EGF-R2A mutants identified a role for the 3′ extended AU pentamer, but not the 5′ extended pentamer, in binding proteins. These EGF-R mRNA-binding proteins were present in multiple human breast and prostate cancer cell lines. In summary, these data demonstrate a central role for mRNA stabilization in the control of EGF-R gene expression in breast cancer cells. EGF-R mRNA contains a novel complex AU-rich 260-nt cis-acting destabilizing element in the 3′-UTR that is bound by specific and EGF-regulatedtrans-acting factors. Furthermore, the 3′ extended AU pentamer of EGF-R2A plays a central role in regulating EGF-R mRNA stability and the binding of specific RNA-binding proteins. These findings suggest that regulated RNA-protein interactions involving this novel cis-acting element will be a major determinant of EGF-R mRNA stability.

The Evolution of MHC Diversity by Segmental Duplication and Transposition of Retroelements

Re: J Mol Evol (1997) 45(6):599–609. The address of Matthew Bellgard should be Centre for Molecular Immunology and Instrumentation, the University of Western Australia, Perth, and Department of Information Technology, Murdoch University, Murdoch, Western Australia. In the first paragraph of Materials and Methods (p. 600), ‘‘a YAC clone (T109) . . .’’ should read ‘‘. . . a YAC clone (Y109) . . .’’ On p. 607, first paragraph, lines 16 and 17, ‘‘. . . more than 25 mya (Shih et al. 1989)’’ should read as ‘‘. . . more than 25 mya (Shih et al. 1991).’’ On p. 609 of the References, Shih A, Misra R, Rush MG (1989) etc. should be replaced with Shih A, Coutavas EE, Rush MG (1991) Evolutionary implications of primate endogenous retroviruses. Virology 182:495–502. J Mol Evol (1998) 46:734

Establishment of “The Gene Mine”: a resource for rapid identification of complex trait genes

Identification of genes underlying complex traits presents a challenge to which geneticists have responded with many diverse approaches. A common feature of these approaches is that different research groups must, on a case-by-case basis, replicate similar efforts in recruitment, genetic characterization, and analyses. To avoid this expensive “churning,” an alternative approach has been proposed: production of an experimental genetic reference population, the Collaborative Cross, in which both genetic diversity and mapping power are maximized. Since this population consists of inbred mouse strains, further advantages are that it is essentially infinitely reproducible; genetic characterization needs to be performed only once; and the founder strains’ genomes have been or will be sequenced, allowing imputation of allele sequences of all members of the reference population. Here we describe the establishment of such a genetic reference population, which we dub “The Gene Mine.” Over 1000 genetically distinct lines have been established, descended from eight diverse founder strains. Preliminary phenotypic ascertainment of these strains indicates unexpected variability arising from independent assortment of genetic variants. The Gene Mine will be a powerful resource for characterization of essentially any mouse phenotype that has a genetic basis.

Rapid Identification of Major-Effect Genes Using the Collaborative Cross

The Collaborative Cross (CC) was designed to facilitate rapid gene mapping and consists of hundreds of recombinant inbred lines descended from eight diverse inbred founder strains. A decade in production, it can now be applied to mapping projects. Here, we provide a proof of principle for rapid identification of major-effect genes using the CC. To do so, we chose coat color traits since the location and identity of many relevant genes are known. We ascertained in 110 CC lines six different coat phenotypes: albino, agouti, black, cinnamon, and chocolate coat colors and the white-belly trait. We developed a pipeline employing modifications of existing mapping tools suitable for analyzing the complex genetic architecture of the CC. Together with analysis of the founders’ genome sequences, mapping was successfully achieved with sufficient resolution to identify the causative genes for five traits. Anticipating the application of the CC to complex traits, we also developed strategies to detect interacting genes, testing joint effects of three loci. Our results illustrate the power of the CC and provide confidence that this resource can be applied to complex traits for detection of both qualitative and quantitative trait loci.

Characterization of Retinal Vascular and Neural Damage in a Novel Model of Diabetic Retinopathy

PURPOSE Diabetic retinopathy (DR) is a major cause of blindness globally. Investigating the underlying mechanisms of DR would be aided by a suitable mouse model that developed key features seen in the human disease, and did so without carrying genetic modifications. This study was undertaken to produce such a model. METHODS Our panel of Collaborative Cross strains was screened for DR-like features after induction of diabetes by intravenous injection with alloxan or streptozotocin. Both flat-mounted whole-retina and histologic sections were studied for the presence of retinal lesions. Progression of DR was also studied by histologic examination of the retinal vascular and neural structure at various time points after diabetes onset. In addition, microarray investigations were conducted on retinas from control and diabetic mice. RESULTS Features of DR such as degenerated pericytes, acellular capillaries, minor vascular proliferation, gliosis of Müller cells, and loss of ganglion cells were noted as early as day 7 in some mice. These lesions became more evident with time. After 21 days of diabetes, severe vascular proliferation, microaneurysms, preretinal damage, increased Müller cell gliosis, and damage to the outer retina were all obvious. Microarray studies found significant differential expression of multiple genes known to be involved in DR. CONCLUSIONS The FOT_FB strain provides a useful model to investigate the pathogenesis of DR and to develop treatments for this vision-threatening disease.

Variable cardiac α-actin (Actc1) expression in early adult skeletal muscle correlates with promoter methylation

Different genes encode the α-actin isoforms that are predominantly expressed in heart and skeletal muscle. Mutations in the skeletal muscle α-actin gene (ACTA1) cause muscle diseases that are mostly lethal in the early postnatal period. We previously demonstrated that the disease phenotype of ACTA1 mouse models could be rescued by transgenic over-expression of cardiac α-actin (ACTC1). ACTC1 is the predominant striated α-actin isoform in the heart but is also expressed in developing skeletal muscle. To develop a translatable therapy, we investigated the genetic regulation of Actc1 expression. Using strains from The Collaborative Cross (CC) genetic resource, we found that Actc1 varies in expression by up to 24-fold in skeletal muscle. We defined significant expression quantitative trait loci (eQTL) associated with early adult Actc1 expression in soleus and heart. eQTL in both heart and soleus mapped to the Actc1 locus and replicate an eQTL mapped for Actc1 in BXD heart and quadriceps. We built on this previous work by analysing genes within the eQTL peak regions to prioritise likely candidates for modifying Actc1 expression. Additionally we interrogated the CC founder haplotype contributions to enable prioritisation of genetic variants for functional analyses. Methylation around the Actc1 transcriptional start site in early adult skeletal muscle negatively correlated with Actc1 expression in a strain-dependent manner, while other marks of regulatory potential (histone modification and chromatin accessibility) were unaltered. This study provides novel insights into the complex genetic regulation of Actc1 expression in early adult skeletal muscles.

Identification of ABCC8 as a contributory gene to impaired early-phase insulin secretion in NZO mice

Type 2 diabetes (T2D) is associated with defective insulin secretion, which in turn contributes to worsening glycaemic control and disease progression. The genetic cause(s) associated with impaired insulin secretion in T2D are not well elucidated. Here we used the polygenic New Zealand Obese (NZO) mouse model, which displays all the cardinal features of T2D including hyperglycaemia to identify genes associated with β-cell dysfunction. A genome-wide scan identified a major quantitative trait locus (QTL) on chromosome 7 associated with defective glucose-mediated insulin secretion. Using congenic strains, the locus was narrowed to two candidate genes encoding the components of the KATP channel: Abcc8 (SUR1) and Kcnj11 (Kir6.2). The NZO Abcc8 allele was associated with a ∼211 bp deletion in its transcript and reduced expression of SUR1. Transgenic NZO mice were generated that expressed the WT Abcc8/Kcnj11 genes and displayed significant improvements in early-phase glucose-mediated insulin secretion and glucose tolerance, confirming Abcc8 as a causative gene. Importantly, we showed that despite improving β-cell function in the NZO transgenic mice, there was no enhancement of insulin sensitivity or body weight. This study provides evidence for a role of Abcc8 in early-phase glucose-mediated insulin secretion and validates this gene as a contributor to β-cell dysfunction in T2D.

Different genetic mechanisms mediate spontaneous versus UVR-induced malignant melanoma

Genetic variation conferring resistance and susceptibility to carcinogen-induced tumorigenesis is frequently studied in mice. We have now turned this to melanoma using the collaborative cross (CC), a resource of mouse strains designed to discover genes for complex diseases. We studied melanoma-prone transgenic progeny across seventy CC genetic backgrounds. We mapped a strong quantitative trait locus for rapid onset spontaneous melanoma onset to Prkdc, a gene involved in detection and repair of DNA damage. In contrast, rapid onset UVR-induced melanoma was linked to the ribosomal subunit gene Rrp15. Ribosome biogenesis was upregulated in skin shortly after UVR exposure, Mechanistically, variation in the “usual suspects” by which UVR may exacerbate melanoma, defective DNA repair, melanocyte proliferation, or inflammatory cell infiltration, did not explain melanoma susceptibility or resistance across the CC. Instead, events occurring soon after exposure, such as dysregulation of ribosome function, which alters many aspects of cellular metabolism, may be important.