GFP-tagged expression and immunohistochemical studies to determine the subcellular localization of the tubby gene family members.

The tubby gene family consists of four members, TUB, TULP1, TULP2 and TULP3, with unknown function. However, a splice junction mutation within the mouse tub gene leads to retinal and cochlear degeneration, as well as maturity onset obesity and insulin resistance. Mutations within human TULP1 have also been shown to co-segregate in several cases of autosomal recessive retinitis pigmentosa (RP) and TULP1 deficiency in mice leads to retinal degeneration. The primary amino acid sequences of the tubby family members do not predict a likely biochemical function. As a first step in defining their function, we present a detailed characterization of the cellular and subcellular localization of the human (TUB) and mouse (tub) homologous gene products. We report the isolation of TUB splice variants which have different subcellular localizations (nuclear versus cytoplasmic) and which define a nuclear localization signal. In addition, using green fluorescent protein (GFP) tags, we observe a nuclear localization for TULP1, similar to TUB splicing forms TUB 561 and TUB 506. Finally, we report tubby expression in mouse brain by in situ hybridization and by immunohistochemistry with polyclonal antibodies. Protein was found in both the hypothalamic satiety centers and in a variety of other CNS structures including the cortex, cerebellum, olfactory bulb and hippocampus. Both nuclear and cytoplasmic signals were detected with a series of independently generated polyclonal antibodies, consistent with the presence of multiple alternatively spliced isoforms within the CNS.

Retinal degeneration but not obesity is observed in null mutants of the tubby-like protein 1 gene.

The tub gene is a member of a small, well conserved neuronal gene family of unknown function. Mutations within this gene lead to early-onset blindness and deafness, as well as late-onset obesity and insulin resistance. To test the hypothesis that mutations within other members of this gene family would lead to similar phenotypes as observed in tubby mice, and hence have similar functional properties, we have generated null mutants of the tubby-like protein ( Tulp ) 1 gene by homologous recombination. Similarly to tubby mice, Tulp1 (-/-)mice exhibit an early-onset retinal degeneration with a progressive, rapid loss of photoreceptors, further supporting the notion that previously identified mutations within the human TULP1 gene are indeed causative of retinitis pigmentosa. However, in contrast to tubby mice, Tulp1 (-/-)mice exhibited normal hearing ability and, surprisingly, normal body weight despite the fact that both TUB and TULP1 are expressed in the same neurons within the hypothalamus in areas known to be involved in feeding behavior and energy homeo stasis. However, TUB and TULP1 show a distinctly different staining pattern in the nucleus of these neurons, perhaps explaining the difference in body weight between the Tulp1 (-/-)and tubby mutant mice.

Retinal degeneration in tulp1-/- mice: vesicular accumulation in the interphotoreceptor matrix.

PURPOSE: The Tulp1 gene is a member of the tubby gene family with unknown function. Mutations in the human TULP1 gene cause autosomal recessive retinitis pigmentosa. To understand the pathogenic mechanism associated with TULP1 mutations and to explore the physiologic function of this protein, we examined tissue distribution of the Tulp1 protein in normal mice and the photoreceptor disease phenotype in Tulp1-ablated mice. METHODS: Tissue distribution of the Tulp1 protein in normal mice was examined by immunoblotting and immunocytochemistry. The disease phenotype in tulp1-/- mice was studied by light and electron microscopy, electroretinography (ERG), and immunocytochemistry. These results were compared with another mouse model of retinal degeneration carrying a rhodopsin mutation. RESULTS: Tulp1 is found exclusively in photoreceptors, localizing predominantly in the inner segments. It is a soluble protein with an apparent molecular weight of approximately 70 kDa. Photoreceptor degeneration developed in tulp1-/- mice, with early involvement of both rods and cones. At the early stage of degeneration, rod and cone opsins, but not peripherin/RDS, exhibited prominent ectopic localization. Electron microscopy revealed massive accumulation of extracellular vesicles surrounding the distal inner segments. CONCLUSIONS: The function of Tulp1 is required to maintain viability of rod and cone photoreceptors. Extracellular vesicular accumulation is not a common phenomenon associated with photoreceptor degeneration but appears to be a distinct ultrastructural feature shared by a small group of retinal disease models. The defect in tulp1-/- mice may be consistent with a loss of polarized transport of nascent opsin to the outer segments.

Cell-specific expression of tubby gene family members (tub, Tulp1,2, and 3) in the retina.

PURPOSE: The family of tubby-like proteins (TULPs), consisting of four family members, are all expressed in-the retina at varying levels. Mutations within two members, tub and TULP1, are known to lead to retinal degeneration in mouse and humans, respectively, suggesting the functional importance of this family of proteins in the retina. Despite a high degree of conservation in the carboxy-terminal region (e.g., putative functional domain of the genes) among family members, they are unable to compensate for one another. The purpose of this study was to provide a rationale for this lack of compensation by investigating the spatial distribution of tubby gene family members in the retina and to investigate the mechanism of photoreceptor cell death in tubby mice. METHODS: In situ hybridization using riboprobes specific for each tubby gene family member and immunohistochemistry for TUB and TULP1 were performed to determine their expression patterns in the retina of tubby and wild-type control mice. The terminal dUTP nick-end labeling (TUNEL) assay was performed to detect apoptotic cells in the retina of tubby and wild-type control mice. RESULTS: tub mRNA was found to be expressed throughout the retina, with highest expression in the ganglion cell layer (GCL) and photoreceptor cells. In contrast, Tulp1 expression was observed only in photoreceptor cells and Tulp3 mRNA was expressed at a moderate level only in the inner nuclear layer (INL) and GCL. The results of the immunohistochemical analysis paralleled those observed in the in situ studies. TUB immunoreactivity was most highly concentrated in the GCL, in the inner and outermost regions of the INL, in the outer plexiform layer (OPL), and in the inner segments of photoreceptor cells. Similarly, TULP1 immunoreactivity was observed in the OPL and inner segments of the photoreceptor cells. No differences in expression at the mRNA or protein level were observed for any of the molecules tested in tubby or wild-type mice. TUNEL-positive cells were detected in the ONL of tubby mice, whereas very few were seen in the same layer of age-matched control mice. CONCLUSIONS: Although all tubby gene family members are expressed in the retina, they each have different cell-specific expression patterns, which may account in part for their inability to compensate for the loss of one family member. The photoreceptor cell death in tubby mice occurs through an apoptotic mechanism, which is known to be the common final outcome of other forms of retinal degeneration.

Genetic modification of hearing in tubby mice: evidence for the existence of a major gene (moth1) which protects tubby mice from hearing loss.

Quantitative trait locus (QTL) analysis of genetic crosses has proven to be a useful tool for identifying loci associated with specific phenotypes and for dissecting genetic components of complex traits. Inclusion of a mutation that interacts epistatically with QTLs in genetic crosses is a unique and potentially powerful method of revealing the function of novel genes and pathways. Although we know that a mutation within the novel tub gene leads to obesity and cochlear and retinal degeneration, the biological function of the gene and the mechanism by which it induces its phenotypes are not known. In the current study, a QTL analysis for auditory brainstem response (ABR) thresholds, which indicates hearing ability, was performed in tubby mice from F(2)intercrosses between C57BL/6J- tub / tub and AKR/J-+/+ F(1)hybrids (AKR intercross) and between C57BL/6J- tub / tub and CAST/Ei.B6- tub / tub F(1)hybrids (CAST intercross). A major QTL, designated asmodifieroftubbyhearing1 ( moth1 ), was identified on chromosome 2 with a LOD score of 33.4 ( P < 10(-33)) in the AKR intercross (181 mice) and of 6.0 ( P < 10(-6)) in the CAST intercross (46 mice). This QTL is responsible for 57 and 43% of ABR threshold variance, respectively, in each strain combination. In addition, a C57BL/6J congenic line carrying a 129/Ola segment encompassing the described QTL region when made homozygous for tubby also exhibits normal hearing ability. We hypothesize that C57BL/6J carries a recessive mutation of the moth1 gene which interacts with the tub mutation to cause hearing loss in tub / tub mice. A moth1 allele from either AKR/J, CAST/Ei or 129/Ola is sufficient to protect C57BL/6J- tub / tub mice from hearing loss.

Advances in the molecular genetics of obesity.

The past year has seen substantial progress in our understanding the molecular mechanisms of bodyweight regulation, particularly in the central and peripheral actions of the leptin and melanocortin signaling pathways (e.g. leptin stimulation of angiogenesis and suppression of cytokine production). Important advances also include the identification of mutations in components of the leptin and melanocortin pathways in human obese families. Expanding from the positional cloning of leptin some five years ago, the mouse continues to be a central focus of study, particularly the way in which different bodyweight-sensing pathways interact in different feeding states.

Molecular characterization of a novel tubby gene family member, TULP3, in mouse and humans.

Tubby and related proteins are derived from a small family of novel genes. The carboxytermini of this family are highly conserved across a number of species including humans, mice, Caenorhabditis elegans, Arabidopsis, rice, and maize. Splicing defects in both tub and another member of the gene family, TULP1 (tubby-like protein 1), lead to phenotypes of retinal degeneration in mice and humans, respectively. We describe here the isolation of the human and mouse homologs of a new family member, TULP3. The cDNAs code for proteins of 442 and 460 amino acids, respectively. The level of identity between the human TULP3 and the mouse homolog is 69%, lower than that observed for the homologs of the other family members (96% for human and mouse TUB), and is higher at the amino- and carboxytermini than in the central region of the protein. Phylogenetically, TULP3 is the family member most closely related to TUB. Also, like TUB, it has a wider pattern of tissue expression than either TULP1 or TULP2. TULP3 is detected at high levels in human RNA from testis, ovaries, thyroid, and spinal chord. Tulp3 is also highly expressed in mouse RNA from eyes and adipose depots, tissues not tested in the human Northern analysis. We also report that TULP3 maps to human chromosome 12p13. The murine homolog, Tulp3, maps to the telomere of mouse chromosome 6.

Recessive mutations in the gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa.

A recessive mutation in the tub gene causes obesity, deafness and retinal degeneration in tubby mice. The tub gene is a member of a family of tubby-like genes (TULPs) that encode proteins of unknown function. Members of this family have been identified in plants, vertebrates and invertebrates. The TULP proteins share a conserved carboxy-terminal region of approximately 200 amino-acid residues. Here we report the analysis of the human gene TULP1, which is expressed specifically in the retina. Upon analysing 162 patients with nonsyndromic recessive retinitis pigmentosa (RP) and 374 simplex cases of RP, we found two who were compound heterozygotes for mutations that cosegregated with disease in the respective families. Three of the mutations are missense changes affecting the conserved C-terminal region; the fourth mutation affects a splice donor site upstream of this region. Our data suggest that mutations in TULP1 are a rare cause of recessive RP and indicate that TULP1 has an essential role in the physiology of photoreceptors.

Molecular characterization of TUB, TULP1, and TULP2, members of the novel tubby gene family and their possible relation to ocular diseases.

Tubby, an autosomal recessive mutation, mapping to mouse chromosome 7, was recently found to be the result of a splicing defect in a novel gene with unknown function. Database searches revealed that sequences corresponding to the C terminus of the tub protein were highly conserved across a number of species including humans, mice, Caenorhabditis elegans, Arabidopsis, rice, and maize, and that tub was a member of a gene family. We describe here, TUB, the human homolog of mouse tub, and two newly characterized family members, TULP1 for tubby like protein 1 and TULP2. These three family members, which differ in the N-terminal half of the protein, share 60-90% amino acid identity across their conserved C-terminal region and have distinct tissue expression patterns. Alternatively spliced transcripts with 5' variable sequences, three of which have been identified for the tubby gene, may mediate tissue specific expression. We also report that TUB, TULP1, and TULP2 map to human chromosomes 11p15.4, 6p21.3, and 19q13.1, respectively. TULP1 and TULP2 map within the minimal intervals identified for retinitis pigmentosa 14 on chromosome 6p21.3 and cone-rod dystrophy on chromosome 19q13.1. TULP1 and TULP2, which are expressed in the retina, make excellent candidates for these ocular diseases as a mutation within the tub gene is known to lead to early progressive retinal degeneration.

A candidate gene for the mouse mutation tubby.

A mutation in the tub gene causes maturity-onset obesity, insulin resistance, and sensory deficits. In contrast to the rapid juvenile-onset weight gain seen in diabetes (db) and obese (ob) mice, obesity in tubby mice develops gradually, and strongly resembles the late-onset obesity seen in the human population. Excessive deposition of adipose tissue eventually leads to a twofold increase of body weight. Tubby mice also suffer retinal degeneration and neurosensory hearing loss. The tripartite character of the tubby phenotype shows striking similarity to human obesity syndromes, such as Alström and Bardet-Biedl. Here we report the identification of a G --> T transversion in a candidate gene that abolishes a donor splice site in the 3' coding region and results in a larger transcript containing the unspliced intron. This alteration is predicted to replace the 44-carboxyterminal amino acids with a 20-amino-acid sequence not found in the wide-type protein. Additionally, a second, prematurely truncated transcript with the unspliced intron is observed in testis messenger RNA and a 2-3-fold increase in brain mRNA is observed in tubby mice compared to B6. The phenotype features of tubby mice may be the result of cellular apoptosis triggered by expression of the mutuated tub gene.

Isolation of conserved sequences from yeast artificial chromosomes by exon amplification.

Exon amplification is a technique designed to solve a central problem in mammalian molecular genetics--specifically, the isolation of genes from large regions of genomic DNA. This technique allows exons to be isolated from genomic DNA following the selective removal of introns by the eukaryotic splicing mechanism. It is a relatively rapid procedure and can theoretically be applied to test the coding potential of very long stretches of genomic DNA. In this paper we describe the application of exon amplification to a mouse yeast artificial chromosome (YAC) 175 kb in length. A number of potential coding sequences were amplified, and two sequences were shown to be conserved across a wide variety of species representing potential genes.

Efficiency and specificity of gene isolation by exon amplification.

Exon amplification is an increasingly popular approach to the identification of transcribed sequences and will complement other strategies to isolate genes. We have used this system to amplify candidate exons from 32 cosmids, including 8 cosmids which span a well characterized 185-kb region of the human major histocompatibility class II region on Chromosome (Chr) 6. We have examined the efficiency, specificity, and reproducibility of the system in isolating exons from genes known to be present on particular cosmids and have determined the nature and frequency of artefact amplifications in routine cosmid screening. We were able to clone at least one exon from 88% (7/8) of all known genes tested (including exons which are differentially spliced) and obtained artefacts from 19% (6/32) of the cosmids tested. Such artefacts generally arise from the amplification of noncoding sequences flanked by regions with high homology to acceptor and donor splice junctions. We show that the exon amplification procedure can be used successfully with a wide variety of cosmids which have different numbers of genes and gene structures and describe several approaches to the characterization of novel exons cloned in this study.

The pharmacy supervisor and the employee pharmacist's job satisfaction.

It seems obvious that satisfaction with one's immediate supervisor would have a significant impact on one's general job satisfaction. However, this relationship has received little attention in the pharmacy literature. This study was designed to determine 1) whether there are differences in job-related satisfaction between pharmacists whose immediate supervisors are pharmacists and those whose supervisors are not pharmacists, and 2) whether the occurrence of conflict between a pharmacist and his or her immediate supervisor is related to the employee pharmacist's job and career satisfaction. The most pronounced finding was the importance of supervisors being pharmacists: satisfaction on five of six satisfaction subscales was related to whether one's supervisor was a pharmacist. Moreover, pharmacists who had the fewest conflicts and disagreements with their supervisors were more satisfied with their choice of pharmacy as a career, their employers, their supervisors, and their jobs.

Evaluation of the experiences of trainees seeking employment after completion of their vocational training.

Two thousand questionnaires were sent to doctors receiving a certificate of prescribed or equivalent experience from the Joint Committee on Postgraduate Training for General Practice over a two-year period. More than 1,400 forms were returned.Seventeen per cent of respondents from 1982 experienced unemployment on completion of their vocational training and this figure had almost doubled by 1983. Some groups of people experienced more problems than others in finding employment but most respondents eventually found a post in which they were happy. Likewise, approval periods and parity varied considerably, and the majority of respondents appeared satisfied with what they achieved.