Novel axon projection after stress and degeneration in the Dscam mutant retina.

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.

Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations.

Mutations in Myo7a cause hereditary deafness in mice and humans. We describe the effects of two mutations, Myo7a(6J) and Myo7a(4626SB), on mechano-electrical transduction in cochlear hair cells. Both mutations result in two major functional abnormalities that would interfere with sound transduction. The hair bundles need to be displaced beyond their physiological operating range for mechanotransducer channels to open. Transducer currents also adapt more strongly than normal to excitatory stimuli. We conclude that myosin VIIA participates in anchoring and holding membrane-bound elements to the actin core of the stereocilium. Myosin VIIA is therefore required for the normal gating of transducer channels.

Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7.

Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder caused by a CAG repeat expansion. To determine the mechanism of neurotoxicity, we produced transgenic mice and observed a cone-rod dystrophy. Nuclear inclusions were present, suggesting that the disease pathway involves the nucleus. When yeast two-hybrid assays indicated that cone-rod homeobox protein (CRX) interacts with ataxin-7, we performed further studies to assess this interaction. We found that ataxin-7 and CRX colocalize and coimmunoprecipitate. We observed that polyglutamine-expanded ataxin-7 can dramatically suppress CRX transactivation. In SCA7 transgenic mice, electrophoretic mobility shift assays indicated reduced CRX binding activity, while RT-PCR analysis detected reductions in CRX-regulated genes. Our results suggest that CRX transcription interference accounts for the retinal degeneration in SCA7 and thus may provide an explanation for how cell-type specificity is achieved in this polyglutamine repeat disease.

Electroretinographic anomalies in mice with mutations in Myo7a, the gene involved in human Usher syndrome type 1B.

PURPOSE: In humans, mutations in the gene encoding myosin VIIa can cause Usher syndrome type 1b (USH1B), a disease characterized by deafness and retinitis pigmentosa. Myosin VIIa is also the gene responsible for the inner ear abnormalities at the shaker1 (sh1) locus in mice. To date, none of the sh1 alleles examined have shown any signs of retinal degeneration. In the present study, electroretinograms (ERGs) were recorded from sh1 mice to determine whether they have any physiological abnormalities. METHODS: ERGs were recorded from mice homozygous for one of nine mutant alleles of Myo7a ranging in age from postnatal day (P)20 to approximately 1 year. All mice were dark adapted for 30 minutes, and all the mutant mice were paired with an appropriately age- and strain-matched control animal. A presumptive null allele of myosin VIIa, Myo7a(4626SB), was used to determine whether mice without myosin VIIa had an increased threshold, as assessed by the light level required to elicit a 15-microV b-wave. RESULTS: At the maximum light intensity used, five of the nine alleles examined had significantly reduced a- and b-wave amplitudes. For example, Myo7a(4626SB) mutant mice had a 20% reduction in a-wave amplitude at the maximum light intensity, and this reduction was the same for mice ranging in age from P20 through 7 months. The b-wave thresholds of the Myo7a(4626SB) mutant mice were not significantly different from those of the control mice. Furthermore, whereas most of the alleles' a-wave implicit times were the same in mutant and control mice, mutant mice with two of the alleles had significantly faster a-wave implicit times. CONCLUSIONS: Mutations in myosin VIIa in mice can lead to decreased ERG amplitudes while threshold remains normal. This is the first report of a physiological anomaly in a mouse model with a mutation in the same gene as involved in USH1B.

Laminin expression in adult and developing retinae: evidence of two novel CNS laminins.

Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known laminin chains within one component of the CNS, the retina. We find seven laminin chains-alpha3, alpha4, alpha5, beta2, beta3, gamma2, and gamma3-outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers-alpha4beta2gamma3 and alpha5beta2gamma3-are present in the retinal matrix. During development, all four of these chains, along with components of laminin 5 (the alpha3, beta3, and gamma2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel laminin heterotrimers in the CNS, which we term here laminin 14 (composed of the alpha4, beta2, and gamma3 chains) and laminin 15 (composed of the alpha5, beta2, and gamma3 chains), and lead us to hypothesize that these laminins, along with laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.

The roles of unconventional myosins in hearing and deafness.

The proper expression and function of several unconventional myosins are necessary for inner-ear function. Mutations in MYO7A and MYO15 cause deafness in humans, and mice. Whereas mutations in Myo6 cause inner-ear abnormalities in mice, as yet no human deafness has been found to the result of mutations in MYO6. In the mammalian inner ear there are at least nine different unconventional myosin isozymes expressed. Myosin 1 beta, VI, VIIa and probably XV are all expressed within a single cell in the inner ear, the hair cell. The myosin isozymes expressed in the hair cell all have unique domains of expression and in some areas, such as the pericuticular necklace, several domains overlap. This suggests that these myosins all have unique functions and that all are individually targeted within the hair cell. The mouse is proving to be a useful model organism for studying both human deafness and elucidating the normal functions of unconventional myosins in vivo.

Disruption of laminin beta2 chain production causes alterations in morphology and function in the CNS.

From the elegant studies of Ramon y Cajal (1909) to the current advances in molecular cloning (e.g., Farber and Danciger, 1997), the retina has served as an ideal model for the entire CNS. We have taken advantage of the well described anatomy, physiology, and molecular biology of the retina to begin to examine the role of the laminins, one component of the extracellular matrix, on the processes of neuronal differentiation and synapse formation in the CNS. We have examined the effect of the deletion of one laminin chain, the beta2 chain, on retinal development. The gross development of retinas from laminin beta2 chain-deficient animals appears normal, and photoreceptors are formed. However, these retinas exhibit several pathologies: laminin beta2 chain-deficient mice display abnormal outer segment elongation, abnormal electroretinograms, and abnormal rod photoreceptor synapses. Morphologically, the outer segments are reduced by 50% in length; the outer plexiform layer of mutant animals is disrupted specifically, because only 7% of observed rod invaginating synapses appear normal, whereas the inner plexiform layer is undisturbed; finally, the rate of apoptosis in the mutant photoreceptor layer is twice that of control mice. Physiologically, the electroretinogram is altered; the amplitude of the b-wave and the slope of the b-wave intensity-response function are both decreased, consistent with synaptic disruption in the outer retina. Together, these results emphasize the prominence of the extracellular matrix and, in particular, the laminins in the development and maintenance of synaptic function and morphogenesis in the CNS.

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.

Identification of the cellular source of laminin beta2 in adult and developing vertebrate retinae.

The interphotoreceptor matrix (IPM) is a specialized extracellular matrix that surrounds the inner and outer segments of photoreceptors. This matrix contains molecules that may be important in directing photoreceptor differentiation and survival. For example, one molecule that we have previously identified as a component of the IPM, laminin beta2 (formerly known as s-laminin), is implicated in the differentiation of rod photoreceptor cells. Developmentally, laminin beta2 is present before rod birth in a position that is consistent with a role in directing rod differentiation; it is found, in both the rat and skate, in the ventricular space that ultimately becomes the IPM. In this study, we identify the source of laminin beta2 in the adult and developing retina. Both immunohistochemistry in the adult skate retina and in situ hybridizations in the adult rat retina reveal that laminin beta2 is produced by Müller cells. In addition, in the skate but not the rat retina, retinal pigment epithelial cells may be an alternative source of laminin beta2. During development, however, laminin beta2 is present before the birth of Müller glial cells; at this stage of development, laminin beta2 RNA is present within the neuroepithelial layer in a pattern that is consistent with its production by neuroepithelial cells.

Developmental expression of laminin beta 2 in rat retina. Further support for a role in rod morphogenesis.

PURPOSE: The authors previously hypothesized that laminin beta 2 (S-laminin) plays a role in directing photoreceptor development. The aim of this study was to examine the temporal and spatial expression pattern of beta 2 laminins in rat retina to test this hypothesis. METHODS: Retinas from Sprague-Dawley rats were harvested on embryonic days (E) 14, 16, and 21, as well as on postnatal days (P) 2, 5, and 10. Cryostat sections were probed with antibodies directed against beta 2 laminin, laminin-1 (alpha 1-beta 1-gamma 1), and von Willebrand factor. RESULTS: At the onset of rod photoreceptor birth (E14), laminin beta 2 surrounds the cells of the retinal pigmented epithelium (RPE) and is present on the apical surface of the retinal neuroepithelium. At E16, laminin beta 2 persists on the apical surface of the neuroepithelium and the subjacent apical surface of the RPE. At birth, laminin beta 2 fills the matrix between the juxtaposed surfaces of the RPE and neuroepithelium; moreover, laminin beta 2 immunoreactivity penetrates the neural retina. Throughout postnatal development, laminin beta 2 immunoreactivity surrounds maturing inner and outer segments. Laminin beta 2 also is found in association with blood vessels in the neural retina itself, as well as with choroidal blood vessels; in both places, it is co-localized with an endothelial marker, von Willebrand factor, and laminin-1. CONCLUSIONS: The spatial and temporal expression of laminin beta 2 is consistent with its hypothesized role in rod development. Laminin beta 2 is in a unique position to interact with mitotically active cells (in early retinal development), uncommitted progenitors (in late embryonic development), developing rods (in early postnatal development), and mature outer segments (throughout adulthood). Together with our earlier functional data, these data support our hypothesis that this molecule is an important component of the interphotoreceptor matrix.

Phosphorolytic error correction during transcription.

Escherichia coli DNA-directed RNA polymerase is shown to contain a novel phosphorolytic error correction activity which removes erroneous nucleotides, as rNDPs, from the 3'-end of the growing transcript. The activity we describe is biochemically similar to polynucleotide phosphorylase (PNP), yet in contrast to PNP is activated by Mn2+. We demonstrate that the activity, which is mediated by Pi, is dependent on the presence of an incorrectly incorporated nucleotide at the leading 3'-end of the transcript. The correction activity we describe exhibits a 4 x 10(4)-fold preference for the excision of incorrect nucleotides from the transcript. These findings suggest the possibility that RNA phosphorolysis may play a critical role in the process of transcriptional proofreading.

The role of RNA polymerase in transcriptional fidelity.

The overall transcription of DNA has previously been demonstrated to proceed at extremely high levels of accuracy. We review the evidence that the process of transcription is subject to proof-reading in the Hopfield sense. In addition, we speculate that the proof-reading activity associated with transcription is subject to cyclical phase transitions. That is, during periods of low processivity associated with initiation, RNA synthesis is relatively imprecise. The transition to the elongation phase of RNA synthesis, characterized by a shift to high processivity, is accompanied by enhanced proof-reading. A model for the damping of transcriptional errors, based on a PPi-mediated processive pyrophosphorolysis reaction, is discussed in terms of pausing during transcription.

Transcriptional proofreading in Escherichia coli.

A novel transcriptional proofreading mechanism associated with the beta-subunit of wild-type RNA polymerase from Escherichia coli is suggested from the following data. The purified holoenzyme contains an NTPase activity which specifically converts noncognate NTPs to their corresponding NDP in a template-dependent manner during in vitro transcription of synthetic single- and double-stranded templates. In contrast, purified enzyme from an rpoB mutant which shows increased transcriptional error lacked template-dependent NTP hydrolytic activity. The NTP hydrolytic activity of wild-type enzyme was critically dependent on the integrity of the initiation complex, and required continued transcriptional elongation. Transcription and translation of the lacZ gene proceeded 17% faster in the mutant than in its wild-type parent. These results are discussed in terms of a proofreading model in which the rate of transcription is limited by proofreading events that involve recognition and hydrolysis of noncognate NTPs before they can be misincorporated into RNA.

Human rhinovirus 3C protease: cloning and expression of an active form in Escherichia coli.

A cDNA encoding the viral protease from the 3C region of human rhinovirus type 14 was expressed in Escherichia coli through the use of a periplasmic secretion vector. The recombinant protease contained an eight amino acid N-terminal extension that enabled its detection by a specific antibody. It was expressed at a level of approximately 1 mg/L of E. coli culture. Biological activity of the protease was assessed in vitro by using a chemically synthesized peptide consisting of a consensus picornavirus protease cleavage site, Arg-Ala-Glu-Leu-Gln-Gly-Pro-Tyr-Asp-Glu. The peptide was cleaved by the recombinant protease at the Gln-Gly bond, generating the product peptides Arg-Ala-Glu-Leu-Gln and Gly-Pro-Tyr-Asp-Glu, which could be separated from the substrate peptide by reversed-phase HPLC. An in vitro assay for the rhinovirus 3C protease was developed by observing the rate of disappearance of the substrate peak from chromatograms of the supernatants of digestion mixtures.

Structure and expression of the genes, mcrBDCGA, which encode the subunits of component C of methyl coenzyme M reductase in Methanococcus vannielii.

The genes that encode the alpha, beta, and gamma subunits of component C of methyl coenzyme M reductase (mcrA, mcrB, and mcrG) in Methanococcus vannielii have been cloned and sequenced, and their expression in Escherichia coli has been demonstrated. These genes are organized into a five-gene cluster, mcrBDCGA, which contains two genes, designated mcrC and mcrD, with unknown functions. The mcr genes are separated by very short intergenic regions that contain multiple translation stop codons and strong ribosomebinding sequences. Although the genome of M. vannielii is 69 mol% A+T, there is a very strong preference in the mcrA, mcrB, and mcrG genes for the codon with a C in the wobble position in the codon pairs AA(U) (C) (asparagine), GA(U) (C) (aspartic acid), CA(U) (C) (histidine), AU(U) (C) (isoleucine), UU(U) (C) (phenylalanine), and UA(U) (C) (tyrosine). The mcrC and mcrD genes do not show this codon preference and frequently have U or A in the wobble position. As the codon pairs listed above are likely to be translated by the same tRNA with a G in the first anticodon position, the presence of C in the wobble position might ensure maximum efficiency of translation of transcripts of these very highly expressed genes.

Expression and purification of native human granulocyte-macrophage colony-stimulating factor from an Escherichia coli secretion vector.

The human granulocyte-macrophage colony stimulating factor (GM-CSF) was expressed and purified from a high-level Escherichia coli secretion vector. A cDNA fragment encoding mature GM-CSF was fused with the aid of a synthetic oligonucleotide to the E. coli outer membrane signal peptide (ompA) of the secretion expression vector pIN-III-ompA3. The primary construction, designated pLB5001, is under transcriptional control of the tandem lipoprotein promoter (lppP) lactose promoter-operator (lacPO), and is regulated by the lactose repressor. Upon induction, a polypeptide of MW = 14,600 was produced which had GM-CSF activity in a human bone marrow colony assay. The linker sequence between the ompA signal peptide and the amino terminus of the mature GM-CSF was removed by oligonucleotide-directed site-specific mutagenesis to produce GM-CSF with an authentic amino terminus. The resulting construct, designated pLB5001-4, expressed authentic GM-CSF with a specific activity similar to that observed for the pLB5001 specified GM-CSF. Both versions of GM-CSF were associated with the membrane fraction after osmotic shock, and were purified to homogeneity by DEAE-Sephacel chromatography, followed by reversed-phase HPLC. Amino acid sequencing from the amino terminus of the purified GM-CSF established that the ompA signal peptide was cleaved at its normal processing site in both cases.

Expression of coliphage T7 in aging anucleate minicells of Escherichia coli.

Anucleate minicells produced by a mutated strain of Escherichia coli remain metabolically active for up to 48 h at 37 degrees C. Minicells of increasing age have been infected with the coliphage T7. Infection results in the onset of transcription and translation producing T7 encoded polypeptides. Quantitative and qualitative changes in T7 gene expression result from infection of increasingly old minicells. There is no detectable increase in the frequency of error occurrence in the synthesis of T7 polypeptides in infected old minicells as compared to infected young minicells.

Mistranslation in bacteriophage-infected anucleate minicells of Escherichia coli: a test for error propagation.

The theory of error propagation proposes that errors occurring during expression of the genetic code lead to increased levels of error occurrence in successive generations. A model system for testing error propagation in bacteriophage T7 infected anucleate minicells of Escherichia coli is described. Errors in translation were were stimulated by addition of gentamicin to phage infected minicells, and the error frequency based on the illegitimate incorporation of L-[35S] cysteine into the T7 0.3 gene protein calculated to be on the order of 1 error per 10 000 codons translated. Errors in the synthesis of T7 early gene products have also been detected as suppression of a UAG nonsense codon in gene 1 of the T7 DNA-dependent RNA polymerase, and as increased charge heterogeneity in the gene 1.3 product (DNA ligase). The question of error propagation has been addressed by infecting minicells with a mutant of T7 containing nonsense mutations in the early gene 1 and late gene 16. Results demonstrate that a T7 DNA-dependent RNA polymerase containing misincorporated amino acids is unable, by mistranscription, to suppress a UAG nonsense codon located in the late T7 gene 16.