Induction of heat shock proteins by hyperthermia and noise overstimulation in hsf1 -/- mice.

J Assoc Res Otolaryngol. 2012 Feb;13(1):29-37

Authors: Gong TW, Fairfield DA, Fullarton L, Dolan DF, Altschuler RA, Kohrman DC, Lomax MI

Abstract
Diverse cellular and environmental stresses can activate the heat shock response, an evolutionarily conserved mechanism to protect proteins from denaturation. Stressors activate heat shock transcription factor 1 (HSF1), which binds to heat shock elements in the genes for heat shock proteins, leading to rapid induction of these important molecular chaperones. Both heat and noise stress are known to activate the heat shock response in the cochlea and protect it from subsequent noise trauma. However, the contribution of HSF1 to induction of heat shock proteins following noise trauma has not been investigated at the molecular level. We evaluated the role of HSF1 in the cochlea following noise stress by examining induction of heat shock proteins in Hsf1 ( +/- ) control and Hsf1 ( -/- ) mice. Heat stress rapidly induced expression of Hsp25, Hsp47, Hsp70.1, Hsp70.3, Hsp84, Hsp86, and Hsp110 in the cochleae of wild-type and Hsf1 ( +/- ) mice, but not in Hsf1 ( -/- ) mice, confirming the essential role of HSF1 in mediating the heat shock response. Exposure to broadband noise (2-20 kHz) at 106 dB SPL for 2 h produced partial hearing loss. Maximal induction of heat shock proteins occurred 4 h after the noise. In comparison to heat stress, noise stress resulted in lower induced levels of Hsp25, Hsp70.1, Hsp70.3, Hsp86, and Hsp110 in Hsf1 ( +/- ) mice. Induction of these heat shock proteins was attenuated, but not completely eliminated, in Hsf1 ( -/- ) mice. These same noise exposure conditions induced genes for several immediate early transcription factors and maximum induction occurred earlier than for heat shock proteins. Thus, additional signaling pathways and transcriptional regulators that are activated by noise probably contribute to induction of heat shock proteins in the cochlea.

PMID: 21932106 [PubMed - indexed for MEDLINE]

Hsp70 inhibits aminoglycoside-induced hair cell death and is necessary for the protective effect of heat shock.

J Assoc Res Otolaryngol. 2008 Sep;9(3):277-89

Authors: Taleb M, Brandon CS, Lee FS, Lomax MI, Dillmann WH, Cunningham LL

Abstract
Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) is a highly conserved stress response that can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in a robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In our system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Therefore, we have begun to examine the role of Hsp70 in mediating the protective effect of heat shock. To determine whether Hsp70 is necessary for the protective effect of heat shock against aminoglycoside-induced hair cell death, we utilized utricles from Hsp70.1/3 (-/-) mice. While heat shock inhibited gentamicin-induced hair cell death in wild-type utricles, utricles from Hsp70.1/3 (-/-) mice were not protected. In addition, we have examined the role of the major heat shock transcription factor, Hsf1, in mediating the protective effect of heat shock. Utricles from Hsf1 (-/-) mice and wild-type littermates were exposed to heat shock followed by gentamicin. The protective effect of heat shock on aminoglycoside-induced hair cell death was only observed in wild-type mice and not in Hsf1 (-/-) mice. To determine whether Hsp70 is sufficient to protect hair cells, we have utilized transgenic mice that constitutively overexpress Hsp70. Utricles from Hsp70-overexpressing mice and wild-type littermates were cultured in the presence of varying neomycin concentrations for 24 h. The Hsp70-overexpressing utricles were significantly protected against neomycin-induced hair cell death at moderate to high doses of neomycin. This protective effect was achieved without a heat shock. Taken together, these data indicate that Hsp70 and Hsf1 are each necessary for the protective effect of heat shock against aminoglycoside-induced death. Furthermore, overexpression of Hsp70 alone significantly inhibits aminoglycoside-induced hair cell death.

PMID: 18512096 [PubMed - indexed for MEDLINE]

Age-related changes in cochlear gene expression in normal and shaker 2 mice.

J Assoc Res Otolaryngol. 2006 Sep;7(3):317-28

Authors: Gong TW, Karolyi IJ, Macdonald J, Beyer L, Raphael Y, Kohrman DC, Camper SA, Lomax MI

Abstract
The vertebrate cochlea is a complex organ optimized for sound transduction. Auditory hair cells, with their precisely arranged stereocilia bundles, transduce sound waves to electrical signals that are transmitted to the brain. Mutations in the unconventional myosin XV cause deafness in both human DFNB3 families and in shaker 2 (sh2) mice as a result of defects in stereocilia. In these mutant mice, hair cells have relatively normal spatial organization of stereocilia bundles but lack the graded, stair-step organization. We used sh2 mice as an experimental model to investigate the molecular consequences of the sh2 mutation in the Myo15 gene. Gene expression profiling with Affymetrix GeneChips in deaf homozygous (sh2/sh2) mice at 3 weeks and 3 months of age, and in age-matched, normal-hearing heterozygotes (+/sh2) identified only a few genes whose expression was affected by genotype, but a large number with age-associated changes in expression in both normal mice and sh2/sh2 homozygotes. Microarray data analyzed using Robust Multiarray Average identified Aim1, Dbi, and Tm4sf3 as genes with increased expression in sh2/sh2 homozygotes. These increases were confirmed by quantitative reverse transcription-polymerase chain reaction. Genes exhibiting altered expression with age encoded collagens and proteins involved in collagen maturation, extracellular matrix, and bone mineralization. These results identified potential cellular pathways associated with myosin XV defects, and age-associated molecular events that are likely to be involved in maturation of the cochlea and auditory function.

PMID: 16794912 [PubMed - indexed for MEDLINE]

Heat shock factor 1-deficient mice exhibit decreased recovery of hearing following noise overstimulation.

J Neurosci Res. 2005 Aug 15;81(4):589-96

Authors: Fairfield DA, Lomax MI, Dootz GA, Chen S, Galecki AT, Benjamin IJ, Dolan DF, Altschuler RA

Abstract
Heat shock proteins (Hsps) can enhance cell survival in response to stress. Heat shock factor 1 (Hsf1) is the major transcription factor that regulates stress-inducible Hsp expression. We previously demonstrated the presence of Hsf1 in the rodent cochlea and also demonstrated that a heat shock known to precondition the cochlea against noise trauma results in Hsf1 activation in the rodent cochlea. In the present study, we used an Hsf1-deficient (Hsf1-/- mouse model to determine whether eliminating the Hsf1-dependent stress pathway would influence hearing loss and/or recovery from a moderate-intensity noise. Hsf1-/- mice and their normal littermates (Hsf1+/+) were exposed to a 98-dB, broadband (2-20 kHz) noise for 2 hr, and auditory brainstem response thresholds were measured at three frequencies (4, 12, and 20 kHz) 3 hr, 3 days, and 2 weeks after noise. Hsf1-/- mice had greater hearing loss than Hsf1+/+ mice, with significant differences in recovery observed at all frequencies tested by 2 weeks after noise. Increased outer hair cell loss was also observed in Hsf1-/- mice following noise. These studies provide evidence for the importance of Hsf1 in cochlear protection, recovery, and/or repair following noise overstimulation.

PMID: 15952177 [PubMed - indexed for MEDLINE]

Deafness-related plasticity in the inferior colliculus: gene expression profiling following removal of peripheral activity.

J Neurochem. 2005 Jun;93(5):1069-86

Authors: Holt AG, Asako M, Lomax CA, MacDonald JW, Tong L, Lomax MI, Altschuler RA

Abstract
The inferior colliculus (IC) is a major center of integration in the ascending as well as descending auditory pathways, where both excitatory and inhibitory amino acid neurotransmitters play a key role. When normal input to the auditory system is decreased, the balance between excitation and inhibition in the IC is disturbed. We examined global changes in gene expression in the rat IC 3 and 21 days following bilateral deafening, using Affymetrix GeneChip arrays and focused our analysis on changes in expression of neurotransmission-related genes. Over 1400 probe sets in the Affymetrix Rat Genome U34A Array were identified as genes that were differentially expressed. These genes encoded proteins previously reported to change as a consequence of deafness, such as calbindin, as well as proteins not previously reported to be modulated by deafness, such as clathrin. A subset of 19 differentially expressed genes was further examined using quantitative RT-PCR at 3, 21 and 90 days following deafness. These included several GABA, glycine, glutamate receptor and neuropeptide-related genes. Expression of genes for GABA-A receptor subunits beta2, beta3, and gamma2, plus ionotropic glutamate receptor subunits AMPA 2, AMPA 3, and kainate 2, increased at all three times. Expression of glycine receptor alpha1 initially declined and then later increased, while alpha2 increased sharply at 21 days. Glycine receptor alpha3 increased between 3 and 21 days, but decreased at 90 days. Of the neuropeptide-related genes tested with qRT-PCR, tyrosine hydroxylase decreased approximately 50% at all times tested. Serotonin receptor 2C increased at 3, 21, and 90 days. The 5B serotonin receptor decreased at 3 and 21 days and returned to normal by 90 days. Of the genes tested with qRT-PCR, only glycine receptor alpha2 and serotonin receptor 5B returned to normal levels of expression at 90 days. Changes in GABA receptor beta3, GABA receptor gamma2, glutamate receptor 2/3, enkephalin, and tyrosine hydroxylase were further confirmed using immunocytochemistry.

PMID: 15934929 [PubMed - indexed for MEDLINE]

Noise overstimulation induces immediate early genes in the rat cochlea.

Brain Res Mol Brain Res. 2004 Nov 4;130(1-2):134-48

Authors: Cho Y, Gong TW, Kanicki A, Altschuler RA, Lomax MI

Abstract
In mammals, exposure to intense noise produces a permanent hearing loss called permanent threshold shift (PTS), whereas a moderate noise produces only a temporary threshold shift (TTS). Little is known about the molecular responses to such high intensity noise exposures. In this study we used gene arrays to examine the early response to acoustic overstimulation in the rat cochlea. We compared cochlear RNA from noise-exposed rats with RNA from unexposed controls. The intense PTS noise induced several immediate early genes encoding both transcription factors (c-FOS, EGR1, NUR77/TR3) and cytokines (PC3/BTG2, LIF and IP10). In contrast, the TTS noise down-regulated the gene for growth hormone. The response of these genes to different noise intensities was examined by quantitative RT-PCR 2.5 h after the 90-min noise exposure. For most genes, the extent of induction correlates with the intensity of the noise exposure. Three proteins (EGR1, NUR77/TR3, and IP10) were detected in many regions of the unexposed cochlea. After exposure to 120 dB noise, these proteins were present at higher levels or showed extended expression in additional regions of the cochlea. LIF was undetectable in the cochlea of unexposed rats, but could be seen in the organ of Corti and spiral ganglion neurons following noise. NUR77/TR3 was a nuclear protein before noise, but following noise translocated to the cytoplasm. These studies provide new insights into the molecular response to noise overstimulation in the mammalian cochlea.

PMID: 15519684 [PubMed - indexed for MEDLINE]

Subcellular localization of WD40 repeat 1 protein in PC12 rat pheochromocytoma cells.

Neurosci Lett. 2004 Sep 9;367(3):399-403

Authors: Shin DH, Lee E, Chung YH, Mun GH, Park J, Lomax MI, Oh SH

Abstract
The dynamics of actin filament protein is crucial for various physiological processes of the cells. Among the proteins correlating with actin dynamics, a novel 67-kDa WD40 repeat protein 1 (WDR1) was the vertebrate homologue of actin-interacting protein 1 (Aip1). Even though previous studies have provided the clues on the function of WDR1 in specific organs under pathological conditions, the exact subcellular localization of WDR1 is not known. Therefore, in the present study, we undertook to determine the distribution of WDR1 within PC12 pheochromocytoma cells (PC12 cells) using light and electron microscopic techniques. Double immunocytochemistry clearly showed that WDR1 immunoreactivities (IRs) were co-localized with anti-actin antibody, suggesting the involvement of WDR1 in actin dynamics. WDR1 immunoreactivities (IRs) in PC12 cells showed different distribution patterns as nerve growth factor (NGF) concentrations varied. During active proliferation, the distribution of WDR1 IRs seemed to be similar to those found in cortical actin patches, whereas WDR1 IR was observed in cytoplasmic actin cables after PC12 cells were induced to differentiate by treating with NGF. Though further studies are necessary to determine the function of WDR1, the current data represents a first step towards the in vitro study of WDR1 protein.

PMID: 15337274 [PubMed - indexed for MEDLINE]

Identification and characterization of choline transporter-like protein 2, an inner ear glycoprotein of 68 and 72 kDa that is the target of antibody-induced hearing loss.

J Neurosci. 2004 Feb 18;24(7):1772-9

Authors: Nair TS, Kozma KE, Hoefling NL, Kommareddi PK, Ueda Y, Gong TW, Lomax MI, Lansford CD, Telian SA, Satar B, Arts HA, El-Kashlan HK, Berryhill WE, Raphael Y, Carey TE

Abstract
The Kresge Hearing Research Institute-3 (KHRI-3) antibody binds to a guinea pig inner ear supporting cell antigen (IESCA) and causes hearing loss. To gain insight into the mechanism of antibody-induced hearing loss, we used antibody immunoaffinity purification to isolate the IESCA, which was then sequenced by mass spectroscopy, revealing 10 guinea pig peptides identical to sequences in human choline transporter-like protein 2 (CTL2). Full-length CTL2 cDNA sequenced from guinea pig inner ear has 85.9% identity with the human cDNA. Consistent with its expression on the surface of supporting cells in the inner ear, CTL2 contains 10 predicted membrane-spanning regions with multiple N-glycosylation sites. The 68 and 72 kDa molecular forms of inner ear CTL2 are distinguished by sialic acid modification of the carbohydrate. The KHRI-3 antibody binds to an N-linked carbohydrate on CTL2 and presumably damages the organ of Corti by blocking the transporter function of this molecule. CTL2 mRNA and protein are abundantly expressed in human inner ear. Sera from patients with autoimmune hearing loss bind to guinea pig inner ear with the same pattern as CTL2 antibodies. Thus, CTL2 is a possible target of autoimmune hearing loss in humans.

PMID: 14973250 [PubMed - indexed for MEDLINE]

Induction of heat shock protein 32 (Hsp32) in the rat cochlea following hyperthermia.

Hear Res. 2004 Feb;188(1-2):1-11

Authors: Fairfield DA, Kanicki AC, Lomax MI, Altschuler RA

Abstract
The genes for heat shock proteins (Hsps) can be upregulated in response to cellular trauma, resulting in enhanced cell survival and protection. Hsp32, also known as heme oxygenase 1, catalyzes the degradation of heme to produce carbon monoxide and bilirubin, which play a variety of cytoprotective functions at physiological concentrations, and iron, which is rapidly sequestered by the iron-binding protein ferritin. In the present study we examined the expression and localization of Hsp32 in the rat cochlea after heat shock using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. Low levels of constitutive Hsp32 expression were observed in the normal rat cochlea by RT-PCR and Western blot. Hsp32 mRNA (messenger RNA) was present at higher levels in a subfraction containing sensorineural epithelium and lateral wall than in a subfraction containing modiolus. Western blot revealed that Hsp32 protein levels increase in the rat cochlea following heat shock. Immunocytochemistry showed scattered staining of outer hair cells in the organ of Corti of normal untreated rats. Following heat shock Hsp32 is upregulated in outer hair cells and the cells of the stria vascularis. These results suggest a potential role for Hsp32 as a component of the oxidative stress response pathway in the rat cochlea.

PMID: 14759565 [PubMed - indexed for MEDLINE]

Pathways for protection from noise induced hearing loss.

Noise Health. 2003 Jul-Sep;5(20):1-17

Authors: Le Prell CG, Dolan DF, Schacht J, Miller JM, Lomax MI, Altschuler RA

Abstract
There is increasing evidence that at least one function of both the medial and the lateral olivocochlear efferent systems is to provide adjustment of the set point of activity in their postsynaptic target, the outer hair cells and afferent processes, respectively. New results, summarized in this review, suggest that both efferent systems can provide protection from noise through this mechanism. There are also intracellular pathways that can provide protection from noise-induced cellular damage in the cochlea. This review also summarizes new results on the pathways that regulate and react to levels of reactive oxygen species in the cochlea as well as the role of stress pathways for the heat shock proteins and for neurotrophic factors in protection, recovery and repair.

PMID: 14558888 [PubMed - indexed for MEDLINE]

Characterization of the human UBE3B gene: structure, expression, evolution, and alternative splicing.

Genomics. 2003 Aug;82(2):143-52

Authors: Gong TW, Huang L, Warner SJ, Lomax MI

Abstract
E3 ubiquitin ligases target proteins for degradation by adding ubiquitin residues. We characterized full-length cDNAs for human and mouse UBE3B, a novel HECT-domain E3 ligase, and analyzed the structure of human UBE3B on chromosome 12q24.1. Alternative splicing of exon 20 of UBE3B generated two major transcripts. The 5.7-kb mRNA lacked exon 20 and encoded a full-length protein ligase, variant 1 (UBE3B_v1). A second transcript contained a 97-bp insertion encoded by exon 20 that introduced an in-frame stop codon. The predicted protein (UBE3B_v2) would lack the HECT domain and would be nonfunctional, since the HECT domain constitutes the active site for ubiquitin transfer. No alternative splicing was observed in this region of mouse UBE3B. Elimination of the HECT domain by alternative splicing has not been reported in any genes encoding HECT domain ligases and may represent a novel mechanism in regulating intracellular levels of functional HECT-domain ligases.

PMID: 12837265 [PubMed - indexed for MEDLINE]

Differential Gene Expression Following Noise Trauma in Birds and Mammals.

Noise Health. 2001;3(11):19-35

Authors: Lomax MI, Gong TW, Cho Y, Huang L, Oh SH, Adler HJ, Raphael Y, Altschuler RA

Abstract
Acoustic overstimulation has very different outcomes in birds and mammals. When noise exposure kills hair cells in birds, these cells can regenerate and hearing will recover. In mammals, however, the hair cell loss, and resulting hearing loss, is permanent. Changes in gene expression form the basis for important biological processes, including repair, regeneration, and plasticity. We are therefore using a battery of molecular approaches to identify and compare changes in gene expression following noise trauma in birds and mammals. Both differential display and subtractive hybridisation were used to identify genes whose expression increased in the chick basilar papilla immediately following exposure to an octave band noise (118 dB, centre frequency 1.5 kHz) for 4-6 hr. Among those upregulated genes were two involved in actin signalling: the CDC42 gene encoding a Rho GTPase, and WDR1, which encodes a protein involved in actin dynamics. A third gene, UBE3B, encodes an E3 ubiquitin ligase involved in protein turnover. A fourth gene encodes a cystein-rich secreted protein that may interact with calcium channels. To examine the mammalian response, gene microarrays on nylon membranes (Clontech Atlas Gene Arrays) were used to examine global changes in gene expression 30 minutes after TTS (110 dB broadband noise 50% duty cycle) or PTS (125 dB, 100% duty cycle) noise overstimulation (each for 90 minutes) in the rat cochlea. Several genes, including classic immediate early response genes such as c-fos, EGR1/NGFI-A, and NGFI-B, were upregulated at this early time point following the PTS exposure, but were not upregulated following the TTS exposure.

PMID: 12689446 [PubMed - as supplied by publisher]

Expression of ZIC genes in the development of the chick inner ear and nervous system.

Dev Dyn. 2003 Apr;226(4):702-12

Authors: Warner SJ, Hutson MR, Oh SH, Gerlach-Bank LM, Lomax MI, Barald KF

Abstract
ZIC genes, vertebrate homologues of the Drosophila pair-rule gene odd-paired (opa), function in embryonic pattern formation, in the early stages of central nervous system neurogenesis and in cerebellar maturation. Mouse Zic genes are expressed in restricted, and in some cases overlapping, patterns during development, particularly in the central and peripheral nervous systems. We identified chick ZIC2 in a differential display analysis of the auditory system designed to find genes up-regulated after noise trauma. In this study, we examined the expression of chick ZIC1, ZIC2, and ZIC3 by in situ hybridization in normal inner ear development and in the tissues that influence its development, including the hindbrain, the neural crest, and the periotic mesenchyme. Between Hamburger and Hamilton stages 13 and 24, all three ZIC genes were found in the dorsal periotic mesenchyme adjacent to the developing inner ear. ZIC1 mRNA was expressed in the otocyst epithelium between stages 12 and 24, in some sensory tissue, as well as in a striped pattern in the floorplate of the hindbrain that appears to be complementary to that of Chordin, a gene known to regulate ZIC expression in frogs. Chick ZIC genes are also expressed in the neuroectoderm, paraxial mesenchyme, brain, spinal cord, neural crest, and/or the overlying ectoderm as well as the limb buds. In general, ZIC1 and ZIC2 expression patterns overlapped, although ZIC2 expression was less robust; ZIC3 expression was minimal. These observations suggest that ZIC genes, in addition to their known roles in brain development, may play an important role in the development of the chick inner ear.

PMID: 12666207 [PubMed - indexed for MEDLINE]

Expression of the mouse Macf2 gene during inner ear development.

Brain Res Mol Brain Res. 2002 Sep 30;105(1-2):67-78

Authors: Leonova EV, Lomax MI

Abstract
Plakins, a family of linker proteins that connect cytoskeletal elements to cellular junctions and the extracellular matrix, are primarily responsible for the mechanical properties of cells and tissues. They include desmoplakin, envoplakin, plectin, dystonin/BPAG1, and Kakapo. Mutations in plakins cause several skin, muscular and neurological disorders. Macrophins are a recently discovered subfamily of plakins with binding domains for actin, intermediate filaments and microtubules. Characteristic features of macrophins include variable actin binding domains, a central rod domain containing both plectin and spectrin repeats, and a C-terminus containing EF hands and GAS2/GAR22 domain. We have examined expression of mouse Macf2, encoding macrophin-2, in adult tissues and in the developing, neonatal, and mature inner ear by in situ hybridization. Northern blot analysis identified three large tissue-specific Macf2 transcripts: a 16-kb mRNA in skeletal muscle and heart, a 15-kb mRNA in brain, and a 9-kb mRNA in RNA from ovary plus uterus. In situ hybridization of the developing mouse inner ear indicated that Macf2 is expressed in the otocyst at day 12.5, in the sensory epithelium by embryonic day 16.5, and in both inner and outer hair cells by day 16.5. Macf2 is expressed in the bodies of both sensory and motor neurons in the central and peripheral nervous system, including the auditory pathway. The Macf2 protein could be involved in the regulation of cytoskeletal connections to cellular junctions and play an important structural role in organs, such as the inner ear, that are subjected to strong mechanical forces.

PMID: 12399109 [PubMed - indexed for MEDLINE]

Expression and localization of heat shock factor (Hsf) 1 in the rodent cochlea.

Hear Res. 2002 Nov;173(1-2):109-18

Authors: Fairfield DA, Kanicki AC, Lomax MI, Altschuler RA

Abstract
Activation of heat shock factors (Hsfs) is one of the potential mechanisms for regulating the transcription of the heat shock proteins (Hsps) and certain other stress-responsive genes. Reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry were used to examine the expression and localization of Hsf1, the stress-responsive member of the Hsf family, in the rat and mouse cochlea. Cerebellum was used as a positive control. Semi-quantitative RT-PCR of cochlear RNA revealed that Hsf1 was more highly expressed in a subfraction containing sensorineural epithelium and lateral wall than in a subfraction containing modiolus, with the alpha splice form predominant over the beta in both subfractions. Immunocytochemistry showed selective staining in the rodent cochlea. Hsf1 immunostaining was found in the nuclei of inner and outer hair cells in the organ of Corti, spiral ganglion cells in the modiolus, and cells in the marginal and intermediate layers of the stria vascularis. This is largely consistent with where Hsp70 induction is reported. Hsf1 activation following heat shock was examined by Western blot. Hyperthermia resulted in stress-induced Hsf1 hyperphosphorylation in cochlea as well as cerebellum. This hyperphosphorylation as well as the correlation of its localization with Hsp70 induction supports a role for Hsf1 in the cochlear stress response.

PMID: 12372640 [PubMed - indexed for MEDLINE]

WDR1 colocalizes with ADF and actin in the normal and noise-damaged chick cochlea.

J Comp Neurol. 2002 Jul 8;448(4):399-409

Authors: Oh SH, Adler HJ, Raphael Y, Lomax MI

Abstract
Auditory hair cells of birds, unlike hair cells in the mammalian organ of Corti, can regenerate following sound-induced loss. We have identified several genes that are upregulated following such an insult. One gene, WDR1, encodes the vertebrate homologue of actin-interacting protein 1, which interacts with actin depolymerization factor (ADF) to enhance the rate of actin filament cleavage. We examined WDR1 expression in the developing, mature, and noise-damaged chick cochlea by in situ hybridization and immunocytochemistry. In the mature cochlea, WDR1 mRNA was detected in hair cells, homogene cells, and cuboidal cells, all of which contain high levels of F-actin. In the developing inner ear, WDR1 mRNA was detected in homogene cells and cuboidal cells by embryonic day 7, in the undifferentiated sensory epithelium by day 9, and in hair cells at embryonic day 16. We also demonstrated colocalization of WDR1, ADF, and F-actin in all three cell types in the normal and noise-damaged cochlea. Immediately after acoustic overstimulation, WDR1 mRNA was seen in supporting cells. These cells contribute to the structural integrity of the basilar papilla, the maintenance of the ionic barrier at the reticular lamina, and the generation of new hair cells. These results indicate that one of the immediate responses of the supporting cell after noise exposure is to induce WDR1 gene expression and thus to increase the rate of actin filament turnover. These results suggest that WDR1 may play a role either in restoring cytoskeletal integrity in supporting cells or in a cell signaling pathway required for regeneration.

PMID: 12115702 [PubMed - indexed for MEDLINE]

Gene expression profiles of the rat cochlea, cochlear nucleus, and inferior colliculus.

J Assoc Res Otolaryngol. 2002 Mar;3(1):54-67

Authors: Cho Y, Gong TW, Stöver T, Lomax MI, Altschuler RA

Abstract
High-throughput DNA microarray technology allows for the assessment of large numbers of genes and can reveal gene expression in a specific region, differential gene expression between regions, as well as changes in gene expression under changing experimental conditions or with a particular disease. The present study used a gene array to profile normal gene expression in the rat whole cochlea, two subregions of the cochlea (modiolar and sensorineural epithelium), and the cochlear nucleus and inferior colliculus of the auditory brainstem. The hippocampus was also assessed as a well-characterized reference tissue. Approximately 40% of the 588 genes on the array showed expression over background. When the criterion for a signal threshold was set conservatively at twice background, the number of genes above the signal threshold ranged from approximately 20% in the cochlea to 30% in the inferior colliculus. While much of the gene expression pattern was expected based on the literature, gene profiles also revealed expression of genes that had not been reported previously. Many genes were expressed in all regions while others were differentially expressed (defined as greater than a twofold difference in expression between regions). A greater number of differentially expressed genes were found when comparing peripheral (cochlear) and central nervous system regions than when comparing the central auditory regions and the hippocampus. Several families of insulin-like growth factor binding proteins, matrix metalloproteinases, and tissue inhibitor of metalloproteinases were among the genes expressed at much higher levels in the cochlea compared with the central nervous system regions.

PMID: 12083724 [PubMed - indexed for MEDLINE]

From gene identification to gene therapy.

Audiol Neurootol. 2002 May-Jun;7(3):161-4

Authors: Kanzaki S, Kawamoto K, Oh SH, Stöver T, Suzuki M, Ishimoto S, Yagi M, Miller JM, Lomax MI, Raphael Y

Abstract
Inner ear disease due to hair cell loss is common, and no restorative treatments for the balance and hearing impairment are currently available. To develop clinical means for enhancing protection and regeneration in the inner ear, it is necessary to understand the molecular basis for hereditary and acquired deafness and vestibular disorders. One approach is to identify and characterize genes that regulate protection or repair in other systems. For that purpose, we have used the differential display assay and compared gene expression between normal and acoustically traumatized inner ears of chicks. Several chick cDNAs that were identified are considered as candidates for roles in the reparative process that follows trauma in the basilar papilla. The mammalian vestibular epithelium has a limited regenerative capability. To identify genes that may participate in the regenerative response, we have used gene arrays profiling, comparing normal to drug-traumatized vestibular epithelia. We identified several genes that are differentially expressed in traumatized vestibular epithelium, including several insulin-like growth factor-I binding proteins. To use this molecular knowledge for enhancing protection and repair in the organ of Corti, it is necessary to overexpress the genes of choice in the inner ear. Using viral-mediated gene transfer, we overexpressed transgenic glial cell line-derived neurotrophic factor and demonstrated a robust protective effect against acoustic and ototoxic inner ear trauma. Future identification of the genes that are important for protection and regeneration, along with improved gene transfer technology, will allow the use of gene therapy for treating hereditary and environmental inner ear disease.

PMID: 12053138 [PubMed - indexed for MEDLINE]

Stress pathways in the rat cochlea and potential for protection from acquired deafness.

Audiol Neurootol. 2002 May-Jun;7(3):152-6

Authors: Altschuler RA, Fairfield D, Cho Y, Leonova E, Benjamin IJ, Miller JM, Lomax MI

Abstract
Noise overstimulation will induce or influence intracellular molecular pathways in the cochlea. One of these is the 'classical' stress response pathway involving heat shock proteins. Hsp70 is induced in the cochlea by a wide variety of stresses including noise, hyperthermia and ototoxic drugs. When a stress that induces Hsp70 is applied to the cochlea, there is protection from a subsequent noise that would normally cause a permanent hearing loss. An upstream regulator of heat shock protein transcription, heat shock factor 1, is expressed in the cochlea and activated by stress. Mice lacking this heat shock factor have reduced recovery from noise-induced hearing loss. The same noise exposure that induces Hsp70 also increases the level of glial cell line-derived neurotrophic factor in the cochlea. Moreover, when this neurotrophic factor is applied into the perilymph of scala tympani prior to a noise exposure there is a significant reduction in hair cell loss and hearing loss. With the potential for activation of multiple pathways in the response to noise, gene microarrays can be useful to examine global gene expression. Initial studies examined differential gene expression immediately following a mild noise exposure (from which there is complete recovery) versus an intense noise (giving profound permanent deafness). Differential expression of several immediate early genes was found following the intense but not the mild noise exposure.

PMID: 12053136 [PubMed - indexed for MEDLINE]

MACF1 gene structure: a hybrid of plectin and dystrophin.

Mamm Genome. 2001 Nov;12(11):852-61

Authors: Gong TW, Besirli CG, Lomax MI

Abstract
Mammalian MACF1 (Macrophin1; previously named ACF7) is a giant cytoskeletal linker protein with three known isoforms that arise by alternative splicing. We isolated a 19.1-kb cDNA encoding a fourth isoform (MACF1-4) with a unique N-terminus. Instead of an N-terminal actin-binding domain found in the other three isoforms, MACF1-4 has eight plectin repeats. The MACF1 gene is located on human Chr 1p32, contains at least 102 exons, spans over 270 kb, and gives rise to four major isoforms with different N-termini. The genomic organization of the actin-binding domain is highly conserved in mammalian genes for both plectin and BPAG1. All eight plectin repeats are encoded by one large exon; this feature is similar to the genomic structure of plectin. The intron positions within spectrin repeats in MACF1 are very similar to those in the dystrophin gene. This demonstrates that MACF1 has characteristic features of genes for two classes of cytoskeletal proteins, i.e., plectin and dystrophin.

PMID: 11845288 [PubMed - indexed for MEDLINE]

Constitutive expression of Hsp27 in the rat cochlea.

Hear Res. 2002 Jan;163(1-2):61-70

Authors: Leonova EV, Fairfield DA, Lomax MI, Altschuler RA

Abstract
Heat shock protein-27 (Hsp27) is known to function as both a stress-inducible molecular chaperone and regulator of actin polymerization. For many cells in the cochlea, actin is part of the cytoskeleton and plays an important role in the maintenance of cochlear function. To understand the molecular processes by which the cochlear actin cytoskeleton is maintained and regulated during normal auditory function, we examined the expression and localization of Hsp27 in the normal rat cochlea. Reverse transcription-polymerase chain reaction and Western blot showed constitutive expression of Hsp27 in the normal rat cochlea. Immunofluorescence microscopy showed Hsp27-like staining is localized to the cuticular plate and lateral wall of outer hair cells. Hsp27-like immunostaining is also found in tension fibroblasts, in the root cells of the spiral limbus and in Reissner's membrane. The presence of Hsp27 in the actin-rich tension fibroblasts and outer hair cells suggests a potential role in the regulation and maintenance of the actin cytoskeleton in these cells. The presence of high levels of constitutive Hsp27 may also provide a mechanism for pre-protecting these cells against environmental stressors.

PMID: 11788200 [PubMed - indexed for MEDLINE]

Glial cell line-derived neurotrophic factor (GDNF) and its receptor complex are expressed in the auditory nerve of the mature rat cochlea.

Hear Res. 2001 May;155(1-2):143-51

Authors: Stöver T, Nam Y, Gong TL, Lomax MI, Altschuler RA

Abstract
Glial cell line-derived neurotrophic factor (GDNF) is a survival factor for many neuronal cell types which signals through a heterodimer receptor consisting of GDNF-family receptor alpha 1 (GFRalpha-1) and Ret (rearranged during transformation). GDNF expression has previously been reported in the inner hair cells of the rat cochlea, with expression of GFRalpha-1 but not Ret in the cell bodies of the auditory nerve (spiral ganglion cells), using in situ hybridization. The present study used reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry to examine GDNF, GFRalpha-1 and Ret in the adult rat auditory nerve. Semi-quantitative RT-PCR showed expression of GDNF and the two receptor components, GFRalpha-1 and Ret, in the modiolar subfraction of the cochlea containing spiral ganglion cells. A shorter mRNA splice variant for GDNF was also detected. Immunocytochemistry showed immunostaining in the modiolus for GDNF, GFRalpha-1 and Ret that was confined to spiral ganglion cells. When RT-PCR expression levels were compared to the expression in the substantia nigra, GFRalpha-1 expression levels were similar, Ret mRNA was lower in the modiolus and GDNF expression was higher in the modiolus. However, when GDNF was further assessed using Western blot, while GDNF protein was found in the modiolus it was at lower levels than in substantia nigra tissue. These results demonstrate that GDNF and both of its receptor components are found in spiral ganglion cells of the adult rat cochlea. Along with the previous report of GDNF in inner hair cells, these new results provide a basis for the role of GDNF as a survival factor for the auditory nerve, as suggested by previous studies.

PMID: 11335084 [PubMed - indexed for MEDLINE]

DFNA25, a novel locus for dominant nonsyndromic hereditary hearing impairment, maps to 12q21-24.

Am J Hum Genet. 2001 Jan;68(1):254-60

Authors: Greene CC, McMillan PM, Barker SE, Kurnool P, Lomax MI, Burmeister M, Lesperance MM

Abstract
Using linkage analysis, we identified a novel dominant locus, DFNA25, for delayed-onset, progressive, high-frequency, nonsyndromic sensorineural hearing loss in a large, multigenerational United States family of Czech descent. On the basis of recombinations in affected individuals, we determined that DFNA25 is located in a 20-cM region of chromosome 12q21-24 between D12S327 (centromeric) and D12S84 (telomeric), with a maximum two-point LOD score of 6.82, at recombination fraction.041, for D12S1030. Candidate genes in this region include ATP2A2, ATP2B1, UBE3B, and VR-OAC. DFNA25 may be the human ortholog of bronx waltzer (bv).

PMID: 11115382 [PubMed - indexed for MEDLINE]

Differential display and gene arrays to examine auditory plasticity.

Hear Res. 2000 Sep;147(1-2):293-302

Authors: Lomax MI, Huang L, Cho Y, Gong TL, Altschuler RA

Abstract
Differential gene expression forms the basis for development, differentiation, regeneration, and plasticity of tissues and organs. We describe two methods to identify differentially expressed genes. Differential display, a PCR-based approach, compares the expression of subsets of genes under two or more conditions. Gene arrays, or DNA microarrays, contain cDNAs from both known genes and novel genes spotted on a solid support (nylon membranes or glass slides). Hybridization of the arrays with RNA isolated from two different experimental conditions allows the simultaneous analysis of large numbers of genes, from hundreds to thousands to whole genomes. Using differential display to examine differential gene expression after noise trauma in the chick basilar papilla, we identified the UBE3B gene that encodes a new member of the E3 ubiquitin ligase family (UBE3B). UBE3B is highly expressed immediately after noise in the lesion, but not in the undamaged ends, of the chick basilar papilla. UBE3B is most similar to a ubiquitin ligase gene from Caenorhabditis elegans, suggesting that this gene has been conserved throughout evolution. We also describe preliminary experiments to profile gene expression in the cochlea and brain with commercially available low density gene arrays on nylon membranes and discuss potential applications of this and DNA microarray technology to the auditory system.

PMID: 10962193 [PubMed - indexed for MEDLINE]

Human COX6A1 gene: promoter analysis, cDNA isolation and expression in the monkey brain.

Gene. 2000 Apr 18;247(1-2):63-75

Authors: Wong-Riley M, Guo A, Bachman NJ, Lomax MI

Abstract
The human COX6A1 gene encodes the ubiquitous isoform of cytochrome c oxidase (COX) subunit VIa (VIa-L), and is located in a CpG island on chromosome 12q24.2. We compared the COX6A1 gene with the published cDNA and several ESTs and concluded that subunit COX VIa-L is synthesized as a preprotein, as are other COX subunits. The same transcription start sites were identified by primer extension analysis of human brain and lymphoblastoid RNA. Analysis of the COX6A1 promoter revealed several conserved sequence elements found in other COX genes, namely binding sites for nuclear respiratory factor 1 (NRF-1), nuclear respiratory factor 2/GA binding protein (NRF-2/GABP), and ying-yang protein 1 (YY1). These conserved elements were shown to bind nuclear proteins from HeLa nuclear extracts. COX6A1 cDNA was isolated from a human brain cDNA library, and the sequence was identical to that of human liver. The expression of this gene was demonstrated by in-situ hybridization in monkey brain sections with our human brain cDNA. Monocular impulse blockade in adult monkeys induced a downregulation of COX6A1 expression in deprived visual neurons, suggesting that this subunit gene is regulated by neuronal activity.

PMID: 10773445 [PubMed - indexed for MEDLINE]

Expression of the GDNF family members and their receptors in the mature rat cochlea.

Brain Res Mol Brain Res. 2000 Mar 10;76(1):25-35

Authors: Stöver T, Gong TL, Cho Y, Altschuler RA, Lomax MI

Abstract
The GDNF family comprises glial cell line-derived neurotrophic factor (GDNF) and the related proteins neurturin, artemin and persephin, which form a subgroup of the TGF-beta superfamily of growth factors. All four neurotrophic factors provide neuronal cell protection and cell survival. GDNF expression was found in the cochlea, and GDNF has been shown to be effective for inner ear protection from drugs and noise-induced insults. As the other members of the GDNF family also provide protective effects on neuronal cells, they may play important roles in the inner ear. We used RT-PCR to examine the expression of GDNF, neurturin, artemin, persephin and their receptors GFRalpha-1, GFRalpha-2, GFRalpha-3 and c-ret in whole rat cochlea as well as in functionally different subfractions (modiolus and sensorineural epithelium/lateral wall) and compared the levels of neurotrophin and receptor mRNAs in the cochlea to those in substantia nigra brain region. Our results demonstrate the expression of all GDNF family members and their receptors in cochlea and substantia nigra. However, the relative levels of mRNA were different for several genes tested in subfractions of the cochlea and/or compared to expression levels in substantia nigra. The presence of mRNA for all four members of the GDNF family and their preferred receptors in the rat cochlea suggests potential functional importance of these neurotrophic factors as protection and survival factors in the inner ear.

PMID: 10719212 [PubMed - indexed for MEDLINE]

A novel mouse kinesin of the UNC-104/KIF1 subfamily encoded by the Kif1b gene.

Gene. 1999 Oct 18;239(1):117-27

Authors: Gong TW, Winnicki RS, Kohrman DC, Lomax MI

Abstract
Kinesin and kinesin-related proteins are microtubule-dependent motor proteins that transport organelles. We have cloned and sequenced a full-length 9924 bp mouse cDNA for a new kinesin of the UNC-104/KIF1 subfamily. Northern blot analysis of mouse RNAs detected high levels of a 10 kb mRNA in brain and eye, but lower levels in other tissues. Human RNA dot-blot analysis detected this mRNA in all tissues examined, although at different levels. The overall structure of the new kinesin (predicted size 204 kDa) was most similar to mouse KIF1A; however, 2.1 kb of the 5' portion of the cDNA were identical to the published sequence for KIF1B (Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., Hirokawa, N., 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-1220). We localized the Kif1b gene to the distal end of mouse Chromosome 4 by haplotype analysis of an interspecific backcross from The Jackson Laboratory. We had previously mapped the gene for the novel kinesin to the same location (Gong, T.-W.L., Burmeister, M., Lomax, M.I., 1996b. The novel gene D4Mille maps to mouse Chromosome 4 and human Chromosome 1p36. Mamm. Genome 7, 790-791). We conclude, therefore, that the Kif1b gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 kDa kinesin, KIF1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, KIF1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1b gene.

PMID: 10571041 [PubMed - indexed for MEDLINE]

The 5' region of the COX4 gene contains a novel overlapping gene, NOC4.

Mamm Genome. 1999 May;10(5):506-12

Authors: Bachman NJ, Wu W, Schmidt TR, Grossman LI, Lomax MI

Abstract
We identified a novel human gene, NOC4 (Neighbor Of COX4), located 5' to COX4, the gene for cytochrome c oxidase subunit IV, on Chr 16q32-ter. Transcripts from this gene were identified among human expressed sequence tags. A full-length, 1.06-kb human retinal NOC4 cDNA encoded a 24-kDa, 210-amino acid hypothetical protein of unknown function. Northern hybridization analysis of human RNAs from various tissues detected NOC4 transcripts of 2.2 and 1.4 kb in all tissues examined, suggesting that NOC4 expression is ubiquitous. Transcription of both the COX4 and NOC4 genes initiates within a 250-bp intergenic promoter and occurs in opposite directions. The bidirectional promoter is G + C-rich, lacks TATA and CCAAT elements, and contains multiple potential binding sites for Sp1 and NRF-2/GABP. Two of the NRF-2/GABP sites are located within 14-bp direct repeats, a conserved feature of mammalian COX4 promoters. The NOC4 and COX4 genes are also linked in the rat, mouse, and bovine genomes. A NOC4-GFP fusion protein is located in both the nucleus and the cytoplasm, including the mitochondria.

PMID: 10337626 [PubMed - indexed for MEDLINE]

A gene upregulated in the acoustically damaged chick basilar papilla encodes a novel WD40 repeat protein.

Genomics. 1999 Feb 15;56(1):59-69

Authors: Adler HJ, Winnicki RS, Gong TW, Lomax MI

Abstract
The chick WDR1 gene is expressed at higher levels in the chick basilar papilla after acoustic overstimulation. The 3.3-kb WDR1 cDNA encodes a novel 67-kDa protein containing nine WD40 repeats, motifs that mediate protein-protein interactions. The predicted WDR1 protein has high sequence identity to WD40-repeat proteins in budding yeast (Saccharomyces cerevisiae), two slime molds (Dictyostelium discoideum and Physarum polycephalum), and the roundworm (Caenorhabditis elegans). The yeast and P. polycephalum proteins bind actin, suggesting that the novel chick protein may be an actin-binding protein. Sequence database comparisons identified mouse and human cDNAs with high sequence identity to the chick WDR1 cDNA. The mouse Wdr1 and human WDR1 proteins showed 95% sequence identity to each other and 86% identity to the chick WDR1 protein. Northern blot analysis of total RNA from the chick basilar papilla after noise trauma revealed increased levels of a 3.1-kb transcript in the lesioned area. The WDR1 gene was mapped to human chromosome 4, between 22 and 24 cM from the telomere of 4p.

PMID: 10036186 [PubMed - indexed for MEDLINE]

The chicken cDNA for ornithine decarboxylase antizyme.

Biochim Biophys Acta. 1998 Mar 4;1396(1):21-6

Authors: Drozdowski B, Gong TW, Lomax MI

Abstract
Two full length avian cDNAs for ornithine decarboxylase antizyme were isolated from a chicken cochlear cDNA library and differed in length through use of alternative poly(A) addition signals. The chick antizyme protein sequence predicted by translational frameshifting of the mRNA is 216 amino acids long and is more similar to Xenopus antizyme than to the mammalian protein.

PMID: 9524208 [PubMed - indexed for MEDLINE]

The Atp1b3 gene for Na,K-ATPase beta 3 subunit maps to mouse chromosome 9, and a related gene, Atp1b3-rs, maps to mouse chromosome 3.

Mamm Genome. 1998 Feb;9(2):171-2

Authors: Besirli CG, Gong TW, Lomax MI

PMID: 9457684 [PubMed - indexed for MEDLINE]

Novel genes expressed in the chick otocyst during development: identification using differential display of RNA.

Int J Dev Neurosci. 1997 Jul;15(4-5):585-94

Authors: Gong TW, Hegeman AD, Shin JJ, Lindberg KH, Barald KF, Lomax MI

Abstract
Differential display of mRNA is a technique that enables the researcher to compare genes expressed in two or more different tissues or in the same tissue or cell under different conditions. The method is based on polymerase chain reaction amplification and comparison of specific subsets of mRNA. We have used this method to clone partial complementary DNAs (cDNAs; amplicons) for genes expressed in the otocyst in order to identify genes that may be involved in development of the inner ear. A full length cDNA was isolated from an embryonic quail head library with an amplicon (KH121) obtained from the otocyst. This avian cDNA encoded a novel, 172-amino acid acidic protein and detected a major transcript of ca 0.8 kb in RNA from chick embryos and several neonatal chick tissues. The full length avian cDNA had high sequence identity to several human cDNAs (expressed sequence tags) from human fetal tissues, including cochlea, brain, liver/spleen and lung, and from placenta. The human homologue of the avian gene encoded a protein that was 183 amino acids long and had 75.6% amino acid sequence identity to the avian protein. These results identified both the avian and human homologues of an evolutionarily conserved gene encoding a small acidic protein of unknown function; however, expression of this gene was not restricted to otocysts.

PMID: 9263035 [PubMed - indexed for MEDLINE]

Complete cDNAs for CDC42 from chicken cochlea and mouse liver.

Biochim Biophys Acta. 1997 Jun 26;1352(3):282-92

Authors: Gong TW, Shin JJ, Burmeister M, Lomax MI

Abstract
CDC42 is a member of the ras superfamily of small GTP-binding proteins that are related through the highly conserved GTP-binding domain and are involved in signal transduction pathways. Two full-length CDC42 cDNAs have been isolated: a 2148-bp chick cochlea cDNA and a 2063-bp mouse liver cDNA. Each encodes a CDC42 protein of 191 amino acids. The avian CDC42 protein differs from the mouse at only one amino acid residue, a Thr for a Ser at position 185. Both CDC42 proteins are more similar to the ubiquitous human isoform originally isolated from placenta than to the isoform isolated from fetal brain. Using a probe from the 3' UTR of the mouse liver CDC42 cDNA, we demonstrated that the mouse gene is expressed in all tissues examined. Southern blot analysis of a mouse inter-specific backcross with this gene-specific probe identified at least three CDC42-like (Cdc42l) genes in the mouse genome. Cdc42l1 was mapped to distal mouse Chromosome 4, near Cappb1. Cdc42l2 mapped more proximal on Chromosome 4, whereas Cdc42l3 mapped to the X Chromosome.

PMID: 9224952 [PubMed - indexed for MEDLINE]

Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms.

Mol Biol Evol. 1997 Jun;14(6):595-601

Authors: Schmidt TR, Jaradat SA, Goodman M, Lomax MI, Grossman LI

Abstract
Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions.

PMID: 9190060 [PubMed - indexed for MEDLINE]

The gene encoding the heart/muscle isoform of cytochrome c oxidase subunit VIII maps to mouse chromosome 7.

Mamm Genome. 1997 Jun;8(6):453-4

Authors: Makris GJ, Lomax MI

PMID: 9166597 [PubMed - indexed for MEDLINE]

Nuclear genes for cytochrome c oxidase.

Biochim Biophys Acta. 1997 May 30;1352(2):174-92

Authors: Grossman LI, Lomax MI

PMID: 9199249 [PubMed - indexed for MEDLINE]

Structure of the human gene (COX6A2) for the heart/muscle isoform of cytochrome c oxidase subunit VIa and its chromosomal location in humans, mice, and cattle.

Genomics. 1997 May 15;42(1):146-51

Authors: Bachman NJ, Riggs PK, Siddiqui N, Makris GJ, Womack JE, Lomax MI

Abstract
We have mapped the gene for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa in three mammalian species and isolated the human COX6AH gene (HGMW-approved symbol COX6A2). The bovine gene was mapped by somatic cell hybrid mapping panels to bovine chromosome BTA 25 with 94-95% concordance. The mouse gene (Cox6ah) was mapped using an interspecific backcross panel from the cross (C57BL/6J x Mus spretus)F1 x Mus spretus probed with the mouse COX VIa-H cDNA. Cox6ah was located on distal chromosome 7, between D7Mit8 and D7Mit13. From the regions of known gene conservation among these three species, we predicted that human COX6AH would be located on chromosome 16p. We hybridized a human x rodent mapping panel of somatic cell hybrids with the human cDNA to confirm this assignment. These data taken together indicated that the human COX6AH gene is located on the short arm of chromosome 16 and facilitated the isolation of the human gene from a chromosome 16-enriched library. The human COX6AH gene spans about 1 kb and contains three exons and two small introns. The sequences of the proximal 5' flanking regions of COX6AH genes are highly conserved between human, bovine, and rodent.

PMID: 9177785 [PubMed - indexed for MEDLINE]

Molecular evolution of cytochrome c oxidase subunit IV: evidence for positive selection in simian primates.

J Mol Evol. 1997 May;44(5):477-91

Authors: Wu W, Goodman M, Lomax MI, Grossman LI

Abstract
Cytochrome c oxidase (COX) is a multi-subunit enzyme complex that catalyzes the final step of electron transfer through the respiratory chain on the mitochondrial inner membrane. Up to 13 subunits encoded by both the mitochondrial (subunits I, II, and III) and nuclear genomes occur in eukaryotic organisms ranging from yeast to human. Previously, we observed a high number of amino acid replacements in the human COX IV subunit compared to mouse, rat, and cow orthologues. Here we examined COX IV evolution in the two groups of anthropoid primates, the catarrhines (hominoids, cercopithecoids) and platyrrhines (ceboids), as well as one prosimian primate (lorisiform), by sequencing PCR-amplified portions of functional COX4 genes from genomic DNAs. Phylogenetic analysis of the COX4 sequence data revealed that accelerated nonsynonymous substitution rates were evident in the early evolution of both catarrhines and, to a lesser extent, platyrrhines. These accelerated rates were followed later by decelerated rates, suggesting that positive selection for adaptive amino acid replacement became purifying selection, preserving replacements that had occurred. The evidence for positive selection was especially pronounced along the catarrhine lineage to hominoids in which the nonsynonymous rate was first faster than the synonymous rate, then later much slower. The rates of three types of "neutral DNA" nucleotide substitutions (synonymous substitutions, pseudogene nucleotide substitutions, and intron nucleotide substitutions) are similar and are consistent with previous observations of a slower rate of such substitutions in the nuclear genomes of hominoids than in the nuclear genomes of other primate and mammalian lineages.

PMID: 9115172 [PubMed - indexed for MEDLINE]

Expression of mal is associated with urothelial differentiation in vitro: identification by differential display reverse-transcriptase polymerase chain reaction.

Differentiation. 1997 Feb;61(3):177-85

Authors: Liebert M, Hubbel A, Chung M, Wedemeyer G, Lomax MI, Hegeman A, Yuan TY, Brozovich M, Wheelock MJ, Grossman HB

Abstract
We have developed an in vitro urothelial differentiation model. In this model, differentiated urothelial cells assemble desmosomes and E-cadherin at cell-cell junctions and stratify and show antigenic and functional evidence for tight junctions. Using this urothelial differentiation model with the differential display reverse-transcriptase polymerase chain reaction (ddRT-PCR), we identified two independently isolated gene fragments that showed near identity with the reported sequence for a human cDNA clone named mal. Differential expression of mal mRNA during urothelial differentiation was confirmed by RT-PCR using two other sets of PCR primers. Furthermore, uncultured urothelial cells from tissues also express mal mRNA, as indicated by RT-PCR. Mal was originally identified in a subtracted cDNA library as a human T-cell differentiation-associated gene and was thought to be T-cell specific. Our results identify mal as a gene also expressed in urothelial cells during differentiation and demonstrate the power of ddRT-PCR for analysis of gene expression under these controlled conditions.

PMID: 9084136 [PubMed - indexed for MEDLINE]

Novel beta 3 isoform of the Na,K-ATPase beta subunit from mouse retina.

Biochim Biophys Acta. 1997 Jan 3;1350(1):21-6

Authors: Besirli CG, Gong TW, Lomax MI

Abstract
We isolated a full-length cDNA encoding a novel 278 amino acid beta subunit of Na,K-ATPase from a mouse retinal cDNA library. The highest sequence identity was to known beta 3 isoforms, identifying the protein as the mouse beta 3 subunit of Na,K-ATPase. Two transcripts, 1.75 kb and 2.1 kb, probably arise from use of alternative poly(A) addition signals.

PMID: 9003452 [PubMed - indexed for MEDLINE]

Beta 4 integrin transfection of UM-UC-2 (human bladder carcinoma) cells: stable expression of a spontaneous cytoplasmic truncation mutant with rapid loss of clones expressing intact beta 4.

Cancer Res. 1997 Jan 1;57(1):38-42

Authors: Kim SY, Bachman NJ, Nair TS, Goldsmith S, Liebert M, Grossman HB, Lomax MI, Carey TE

Abstract
The alpha 6 beta 4 integrin is a component of the hemidesmosome, the anchoring structure in the basal membrane of epithelial cells. alpha 6 beta 4 expression is frequently altered in neoplastic cells. It is sometimes lost and sometimes overexpressed, which suggests that disruption of normal function is involved in neoplastic transformation. To examine the effect of this integrin on the growth and behavior of malignant cells that have lost beta 4, we transfected a full-length beta 4 cDNA into the UM-UC-2 cell line that expresses alpha 6 but not beta 4. Although large numbers of clones were obtained when a control vector was used in the transfection, only 12 clones could be isolated that expressed beta 4. Of these, only two beta 4-positive clones, clones 8 and 11, persisted long enough for further study. Clone 8 cells initially expressed beta 4, but within 2 weeks, all positive cells were lost from the culture. Clone 11 persisted in culture and retained strong surface expression of alpha 6 beta 4. Biochemical analysis and Western blotting revealed that this clone contained a truncated form of beta 4 that had lost the distal cytoplasmic domain. We conclude that expression of wild-type beta 4 in UM-UC-2 inhibits cell growth, presumably by an integrin-mediated signaling pathway. Clone 11 escaped from normal signaling because the cytoplasmic domain, a region essential for basal polar localization, was lost. The alpha 6 beta 4 integrin appears to have tumor suppressor activity in epithelial tumors.

PMID: 8988037 [PubMed - indexed for MEDLINE]

The novel gene D4Mil1e maps to mouse chromosome 4 and human chromosome 1p36.

Mamm Genome. 1996 Oct;7(10):790-1

Authors: Gong TL, Burmeister M, Lomax MI

PMID: 8854876 [PubMed - indexed for MEDLINE]

The gene encoding subunit IV of cytochrome c oxidase maps to mouse chromosome 8.

Mamm Genome. 1996 Oct;7(10):789-90

Authors: Makris GJ, Samuelson LC, Lomax MI

PMID: 8854875 [PubMed - indexed for MEDLINE]

Sequence of the cDNA for the liver/non-muscle isoform of mouse cytochrome-c oxidase subunit VIII.

Biochim Biophys Acta. 1996 Sep 11;1308(3):197-200

Authors: Makris GJ, Lomax MI

Abstract
We have isolated and sequenced the cDNA for the liver (L) or non-muscle isoform of mouse cytochrome-c oxidase subunit VIII (COX VIII-L). Comparison of deduced COX VIII-L protein sequences from three mammalian species indicated that the human gene has sustained more amino acid replacement substitutions than either the mouse or the cow. The most highly conserved regions of this subunit are the N-terminal presequence and the C-terminal domain of the mature protein.

PMID: 8809110 [PubMed - indexed for MEDLINE]

Phylogenetic footprinting of the human cytochrome c oxidase subunit VB promoter.

Arch Biochem Biophys. 1996 Sep 1;333(1):152-62

Authors: Bachman NJ, Yang TL, Dasen JS, Ernst RE, Lomax MI

Abstract
The human COX5B gene encodes subunit Vb of cytochrome c oxidase (COX). COX Vb is 1 of the 10 subunits of the mitochondrial COX complex encoded by a nuclear gene. We have defined a region in the human COX5B promoter essential for gene expression and shown by phylogenetic footprinting of 11 primate COX5B promoters that many cis-regulatory elements in this region are evolutionarily conserved. The transcription start site of human COX5B was mapped 58 bp upstream of the initiation Met codon by primer extension using a thermostable reverse transcriptase. A 475-bp region (-456 to +20) of the human COX5B gene was shown to function as a promoter for the chloramphenicol acetyl transferase (CAT) gene in expression vectors when transfected into HeLa cells. The human COX5B gene is located in a CpG island and contains several potential binding sites for the transcription factor Sp1, but no consensus TATA box element. Several sequence elements associated with the transcriptional regulation of respiratory genes were also found in the promoter and 5' flanking region, including a single NRF-1 site and two 9-bp direct repeats containing binding sites for ets-domain proteins, such as NRF-2/GABP. Many features of the human COX5B promoter are conserved in the COX5B promoters of primates, in particular, the presence of a single binding site for NRF-1 and multiple sites for Sp1 and NRF-2/GABP. Electrophoretic mobility shift assays demonstrate that the conserved NRF-1 site in primate COX5B promoters is specifically recognized by a factor present in HeLa nuclear extracts. Phylogenetic footprinting has identified additional conserved elements that may also function as binding sites for regulatory factors.

PMID: 8806766 [PubMed - indexed for MEDLINE]

Identification of genes expressed after noise exposure in the chick basilar papilla.

Hear Res. 1996 Jul;96(1-2):20-32

Authors: Gong TW, Hegeman AD, Shin JJ, Adler HJ, Raphael Y, Lomax MI

Abstract
We used differential display of mRNA, a method based on reverse transcriptase-PCR, to identify genes whose expression increases in response to acoustic trauma in the chick basilar papilla. Identifying these genes would provide insight into processes involved in repair of the damaged epithelium or in hair cell regeneration. We compared mRNA from the basilar papilla of normal chicks, from chicks exposed to an octave band noise (center frequency: 1.5 kHz) presented at 118 dB for 6 h, and from chicks exposed to noise and allowed to recover for 2 days. Thus far, we have identified 70 bands that appear to be differentially displayed on DNA sequencing gels; approximately 40 of these bands have been subcloned and sequenced. DNA sequences were compared with sequences in the GenBank database to identify genes with significant (70-85%) sequence identity to known genes. Chick cDNAs identified included: the parathyroid hormone-related protein, an immediate early gene; the delta-subunit of the neuronal-specific Ca2+/calmodulin-regulated protein kinase II; and the GTP-binding protein CDC42, a member of the ras superfamily of G proteins. A fourth cDNA had 84% sequence identity to an uncharacterized human cDNA (expressed sequence tag), indicating that this is a novel gene. Slot-blot hybridization analysis of these cDNAs probed with labeled DNA generated from mRNA from each experimental group indicated higher levels of mRNA for each of these four genes after noise exposure. These results indicate the potential involvement of both Ca2+/calmodulin-mediated signaling and GTPase cascades in the response to noise damage and during hair cell regeneration in the chick basilar papilla.

PMID: 8817303 [PubMed - indexed for MEDLINE]

Sequence of the cDNA for the heart/muscle isoform of mouse cytochrome c oxidase subunit VIII.

Biochim Biophys Acta. 1995 Apr 4;1261(2):311-4

Authors: Hegeman AD, Brown JS, Lomax MI

Abstract
We have isolated and sequenced cDNAs for the heart/muscle (H) isoform of mouse cytochrome c oxidase subunit VIII (COX VIII-H). The deduced protein sequence enables us to compare the heart/muscle COX VIII isoforms from several species and to determine that the most highly conserved region of this subunit is the C-terminal domain.

PMID: 7711081 [PubMed - indexed for MEDLINE]

Structure and chromosomal location of the bovine gene for the heart muscle isoform of cytochrome c oxidase subunit VIII.

Mamm Genome. 1995 Feb;6(2):118-22

Authors: Lomax MI, Riggs PK, Womack JE

Abstract
We have isolated the bovine COX8H gene for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIII from a library of bovine genomic DNA cloned into lambda EMBL3. Primer extension assays on bovine heart mRNA mapped the 5' ends of COX8H transcripts to a CA dinucleotide 62-bp upstream from the ATG codon. The gene thus spans 1565-bp and comprises two exons and one large intron of 1227 bp. Exon 1 encodes the 5' untranslated region, a 24-amino acid presequence, and the first 13 amino acids of the mature COX VIII-H protein. Exon 2 encodes the remainder of the cDNA: amino acids 14 to 46 plus the 66-bp 3' untranslated region. The exon-intron boundaries matched the consensus splice junction sequences. Two protein polymorphisms were seen: an Ala/Val polymorphism at position -6 in the presequence and the previously noted Lys/Arg polymorphism at residue 7 of the mature protein. A TaqI polymorphism occurs in the intron. The COX8H gene was mapped by bovine x rodent somatic cell hybrid mapping panels to bovine (BTA) Chromosome (Chr) 25 with 100% concordancy. BTA 25 is conserved relative to the long arm of human (HSA) Chr 11, which contains COX8, the gene for the single human COX VIII subunit that is homologous to the liver isoform.

PMID: 7766994 [PubMed - indexed for MEDLINE]

Structural organization of the bovine gene for the heart/muscle isoform of cytochrome c oxidase subunit VIa.

Biochim Biophys Acta. 1993 Jul 18;1174(1):63-71

Authors: Smith EO, Lomax MI

Abstract
The bovine gene for the nuclear-encoded heart/muscle isoform of cytochrome c oxidase subunit VIa (COX6A1) was isolated from a library of bovine genomic DNA in lambda EMBL3 and sequenced. The gene spans 760 bp and comprises three exons and two small introns. Exon 1 encodes a 193 bp 5' untranslated region, a 12 amino acid presequence, and the first 12 amino acids of the mature COX VIa protein. Exon 2 encodes amino acids 13 to 58, and exon 3 amino acids 59 to 85 plus the 35 bp 3' untranslated region. Exons 2 and 3 are separated by a small intron of only 96 bp. All exon-intron boundaries matched the consensus splice junction sequences. COX6A1 transcripts are present in RNA from bovine heart but not brain. Primer extension and ribonuclease protection assays were used to map the 5' ends of COX6A1 transcripts in heart; both methods identified several clusters of transcription initiation sites, indicating that COX6A1 mRNA is heterogeneous at the 5' end. The proximal 5' flanking region is AT-rich and contains potential basal promoter elements, such as TATA and CCAAT boxes, associated with tissue-specific genes. A single consensus binding site for the muscle-specific transcription factor, MyoD1, was also located within this AT-rich region. The distal promoter region contained a perfect AP4 site plus potential binding sites for enhancer elements (NRF-1, Mt1, Mt3, and Mt4) proposed to regulate expression of genes for mitochondrial proteins.

PMID: 7687470 [PubMed - indexed for MEDLINE]

Rapid evolution of the human gene for cytochrome c oxidase subunit IV.

Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5266-70

Authors: Lomax MI, Hewett-Emmett D, Yang TL, Grossman LI

Abstract
We have compared the DNA sequences of nine mammalian genes for cytochrome c oxidase subunit IV (COX4 genes)--four expressed genes (human, bovine, rat, and mouse) and five pseudogenes (human, chimpanzee, orangutan, squirrel monkey, and bovine)--and constructed the sequence of the ancestral mammalian COX4 gene. By analyzing these sequences to determine the pattern and rate of nucleotide substitution in each branch of the evolutionary tree, we deduced that the human gene has evolved rapidly since the origin of the primate pseudogene approximately 41 million years ago, and we discuss the suggestion that this results from coevolution of nuclear and mitochondrial genes for cytochrome c oxidase.

PMID: 1319058 [PubMed - indexed for MEDLINE]

Myosin light chain 1 isoform expression remains constant during ageing in Wistar F455 rats.

Mech Ageing Dev. 1991 Dec 2;61(2):163-72

Authors: Dunathan AL, Lomax MI, Barald KF

Abstract
In order to study muscle gene expression during ageing, we examined both protein and total cellular RNA from Wistar F455 rat soleus and extensor digitorum longus (EDL) muscles at a variety of chronological ages. We found no evidence of the reappearance of the fast protein isoform of myosin light chain 1 [MLC1] in the slow soleus muscle during ageing previously reported by Syrovy and Gutmann, Pflügers Arch., 369 (1977) 85-89. We used both SDS-PAGE analysis of MLC1 proteins and slot blot RNA analysis with a probe specific for rat fast MLC1 mRNA (pC91), and found no changes in fast MLC1 expression during ageing in soleus or EDL muscles from these rats. These results indicate that re-expression of the fast MLC1 isoform is not a universal property of ageing soleus muscle.

PMID: 1726698 [PubMed - indexed for MEDLINE]

Characterization and expression of a cDNA specifying subunit VIIc of bovine cytochrome c oxidase.

Gene. 1991 Aug 15;104(2):211-7

Authors: Aqua MS, Bachman NJ, Lomax MI, Grossman LI

Abstract
We have isolated a cDNA that encodes subunit VIIc of bovine cytochrome c oxidase (COX VIIc). The 325-bp cDNA contains sequences encoding the mature 47-amino acid (aa) polypeptide and a 16-aa presequence. The deduced aa sequence of the processed polypeptide is identical to that of the heart protein determined by aa sequencing. Northern-blot analysis reveals a single 525-nucleotide (nt) transcript in all tissues examined, whose levels vary with the corresponding respiratory activities in different tissues; thus, no evidence for isoforms of COX VIIc is seen in adult tissues. Southern-blot analysis of bovine genomic DNA digested with three different restriction enzymes reveals several bands that hybridize with the cDNA. We present here the sequence of one genomic region that contains a processed gene encoding COX VIIc. The genomic and cDNA nt sequences are 99% identical throughout the 189-bp open reading frame; the deduced aa sequences are identical. The sequence of the genomic clone suggests that the cDNA terminates prematurely at an EcoRI site in the 3'-untranslated region. We have compared COX VIIc cDNAs from cow, human and mouse, and find the presequence similarity among them to be 100% at the aa level.

PMID: 1655579 [PubMed - indexed for MEDLINE]

The cDNA for the heart/muscle isoform of bovine cytochrome c oxidase subunit VIa encodes a presequence.

Biochim Biophys Acta. 1991 Jun 13;1089(2):266-8

Authors: Smith EO, BeMent DM, Grossman LI, Lomax MI

Abstract
We have used mixed oligonucleotide probes to isolate a cDNA for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa (COX VIa-H) from a bovine heart cDNA library in lambda gt10. This cDNA, and a second one isolated upon rescreening, predict a 97 amino acid COX VIa precursor protein comprised of a 12 amino acid, basic presequence plus an 85 residue mature VIa protein. The presence of a presequence contrasts with the rat heart COX VIa cDNA.

PMID: 1647214 [PubMed - indexed for MEDLINE]

Structure of the human cytochrome c oxidase subunit Vb gene and chromosomal mapping of the coding gene and of seven pseudogenes.

Genomics. 1991 May;10(1):1-9

Authors: Lomax MI, Hsieh CL, Darras BT, Francke U

Abstract
Subunit Vb of mammalian cytochrome c oxidase (COX; EC 1.9.3.1) is encoded by a nuclear gene and assembled with the other 12 COX subunits encoded in both mitochondrial and nuclear DNA. We have cloned the gene for human COX subunit Vb (COX5B) and determined the exon-intron structure by both hybridization analysis and DNA sequencing. The gene contains five exons and four introns; the four coding exons span a region of approximately 2.4 kb. The 5' end of the COX5B gene is GC-rich and contains many HpaII sites. Genomic Southern blot analysis of human DNA probed with the human COX Vb cDNA identified eight restriction fragments containing COX Vb-related sequences that were mapped to different chromosomes with panels of human x Chinese hamster somatic cell hybrids. Because only one of these fragments hybridized with a 210-bp probe from intron 4, we conclude that there is a single expressed gene for COX subunit Vb in the human genome. We have mapped this gene to chromosome 2, region cen-q13.

PMID: 1646156 [PubMed - indexed for MEDLINE]

Differential expression of nuclear genes for cytochrome c oxidase during myogenesis.

Muscle Nerve. 1990 Apr;13(4):330-7

Authors: Lomax MI, Coucouvanis E, Schon EA, Barald KF

Abstract
Recent studies of patients with mitochondrial myopathies suggest the existence of both muscle-specific and developmentally regulated isoforms of cytochrome c oxidase (COX), the terminal enzyme complex of the electron transport chain. To investigate the temporal pattern of gene expression of nuclear genes for COX in developing muscle, the steady-state levels of COX mRNA in total RNA from a satellite cell-derived mouse muscle cell line, C2C12, were analyzed and compared with COX mRNA levels in mature rat skeletal muscle. Undifferentiated myoblasts, myotubes just after fusion (early myotubes), and fully differentiated, contractile, striated myotubes (late myotubes) were analyzed for mRNA levels for four of the 10 different nuclear-encoded COX subunits: IV, Vb, Vlc and VIII-liver. Of these, IV, Vb and Vlc are identical in both bovine heart and liver, whereas subunit VIII has heart and liver isoforms. In C2C12 myoblasts, the level of mRNA for subunits IV, Vb, and VIII-liver is equal to or greater than the level in tissues such as brain, skeletal muscle, and liver. As myoblasts fuse and differentiate into myotubes, the levels of mRNA for these subunits undergo radically different changes. Transcripts for subunits IV and Vb accumulate to higher levels during myogenesis. The level of subunit VIII transcripts decreases during myogenesis, providing additional evidence that subunit VIII has tissue-specific isoforms in the rat. Little mRNA for COX Vlc was detected in either the C2C12 cell line or in primary embryonic rat myoblasts or myotubes in culture in spite of high levels in adult skeletal muscles, suggesting that subunit Vlc may have both fetal and adult isoforms in rodents.

PMID: 2162485 [PubMed - indexed for MEDLINE]

Novel use of a chimpanzee pseudogene for chromosomal mapping of human cytochrome c oxidase subunit IV.

Gene. 1990 Feb 14;86(2):209-16

Authors: Lomax MI, Welch MD, Darras BT, Francke U, Grossman LI

Abstract
We have isolated a chimpanzee processed pseudogene for subunit IV of cytochrome c oxidase (COX; EC 1.9.3.1) by screening a chimpanzee genomic library in lambda Charon 32 with a bovine liver cDNA encoding COX subunit IV (COX IV), and localized it to a 1.9-kb HindIII fragment. Southern-blot analysis of genomic DNA from five primates showed that DNAs from human, gorilla, and chimpanzee each contained the 1.9-kb pseudogene fragment, whereas orangutan and pigtail macaque monkey DNA did not. This result clearly indicates that the pseudogene arose before the divergence of the chimpanzee and gorilla from the primate lineage. By screening Chinese hamster x human hybrid panels with the human COX4 cDNA, we have mapped COX4 genes to two human chromosomes, 14 and 16. The 1.9-kb HindIII fragment containing the pseudogene, COX4P1, can be assigned to chromosome 14, and by means of rearranged chromosomes in somatic cell hybrids, to 14q21-qter. Similarly, the functional gene, COX4, has been mapped to 16q22-qter.

PMID: 2157630 [PubMed - indexed for MEDLINE]

Sequence of a cDNA specifying subunit VIIc of human cytochrome c oxidase.

Nucleic Acids Res. 1990 Feb 11;18(3):684

Authors: Koga Y, Fabrizi GM, Mita S, Arnaudo E, Lomax MI, Aqua MS, Grossman LI, Schon EA

PMID: 2155413 [PubMed - indexed for MEDLINE]

Tissue-specific genes for respiratory proteins.

Trends Biochem Sci. 1989 Dec;14(12):501-3

Authors: Lomax MI, Grossman LI

Abstract
All but one of the mitochondrial respiratory complexes are composed of products of both the mitochondrial and the nuclear genomes. The recent isolation of cDNAs for several nuclear-encoded respiratory proteins reveals that some of them are present in at least two forms. Although some of these forms are traditional in differing somewhat in amino acid sequence, a new class, termed silent isoforms, differs in the presequence but contains identical processed proteins. What are the roles of tissue isoforms in oxidative metabolism?

PMID: 2560276 [PubMed - indexed for MEDLINE]

Nucleotide sequence of a cDNA for bovine cytochrome c oxidase subunit VIIc.

Nucleic Acids Res. 1989 Oct 25;17(20):8376

Authors: Aqua MS, Lomax MI, Schon EA, Grossman LI

PMID: 2554257 [PubMed - indexed for MEDLINE]

Sequence of a cDNA specifying subunit VIIa of human cytochrome c oxidase.

Nucleic Acids Res. 1989 Sep 12;17(17):7107

Authors: Fabrizi GM, Rizzuto R, Nakase H, Mita S, Lomax MI, Grossman LI, Schon EA

PMID: 2550906 [PubMed - indexed for MEDLINE]

Nucleotide sequence of a cDNA for bovine cytochrome c oxidase subunit VIIa.

Nucleic Acids Res. 1989 Aug 11;17(15):6410

Authors: Seelan RS, Scheuner D, Lomax MI, Grossman LI

PMID: 2549516 [PubMed - indexed for MEDLINE]

Two bovine genes for cytochrome c oxidase subunit IV: a processed pseudogene and an expressed gene.

Gene. 1987;55(2-3):219-29

Authors: Bachman NJ, Lomax MI, Grossman LI

Abstract
We have isolated and analyzed 17 clones from a bovine genomic library in phage lambda Charon28 probed with a bovine liver cDNA for cytochrome c oxidase subunit IV. Restriction enzyme mapping and Southern analysis indicated that these clones represent only two genomic regions. One region was shown by nucleotide sequencing to contain a subunit IV pseudogene of the processed type. The other class of clones contained the 5' region of a putative expressed gene; the region consists of two exons and two introns, with one exon encoding exclusively the domain representing the presequence present on newly synthesized subunit-IV polypeptides. Genomic Southern analysis indicated that these two clones probably represent the only sequences in the bovine nucleus that share nucleotide sequence identity with the liver subunit IV cDNA when utilizing moderately stringent hybridization conditions.

PMID: 2822541 [PubMed - indexed for MEDLINE]

Isolation of a cDNA clone encoding subunit IV of human cytochrome c oxidase.

Gene. 1987;55(2-3):205-17

Authors: Zeviani M, Nakagawa M, Herbert J, Lomax MI, Grossman LI, Sherbany AA, Miranda AF, DiMauro S, Schon EA

Abstract
We have isolated a full-length human liver cDNA clone specifying the nuclear-encoded subunit IV of the human mitochondrial respiratory chain enzyme, cytochrome c oxidase (COX; EC 1.9.3.1). The human cDNA clone is highly homologous to its bovine counterpart in the coding regions for both the mature polypeptide and the presequence, and the gene is evolving more slowly than that of any of the three mitochondrially encoded COX subunit genes. We find no preliminary evidence for tissue-specific isoforms of COX subunit IV, as Northern analysis of muscle, liver, and HeLa cell RNA shows an identically sized transcript in each cell type.

PMID: 2444497 [PubMed - indexed for MEDLINE]

SPLINT: a cubic spline interpolation program for the analysis of fragment sizes in one-dimensional electrophoresis gels.

Nucleic Acids Res. 1986 Jan 10;14(1):575-81

Authors: Gariepy CE, Lomax MI, Grossman LI

Abstract
SPLINT (SPLine INTerpolation) is a BASIC microcomputer program that uses electrophoretic data on the migration distances of DNA fragments of known size to approximate the relationship between distance moved and DNA fragment size by a piecewise cubic spline function. This function places all standards exactly on the calibration curve and then determines from it the sizes of other fragments in the gel. Special advantages of this program include the use of a digitizer to enter the fragment positions directly from a gel photograph and the optional generation of a graph of the approximating function plus the placement on it of points from any specified gel lanes.

PMID: 3003683 [PubMed - indexed for MEDLINE]

Isolation and characterization of a cDNA clone for bovine cytochrome c oxidase subunit IV.

Proc Natl Acad Sci U S A. 1984 Oct;81(20):6295-9

Authors: Lomax MI, Bachman NJ, Nasoff MS, Caruthers MH, Grossman LI

Abstract
We have isolated a cDNA clone for the precursor to subunit IV of bovine cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). A cDNA library was constructed from poly(A)+ RNA of adult beef liver by insertion of cDNA into the plasmid vector pBR322. Transformants were screened by colony hybridization with two mixtures of [32P]-labeled synthetic oligodeoxyribonucleotides. We screened 20,000 transformants with a mixture of heptadecamers complementary to all 16 possible sequences encoding amino acids 98-103 and obtained two cDNA clones encoding subunit IV amino acid sequences. We determined the DNA sequence of the larger (416 base-pair) insert, which contains the coding sequence for amino acids 1-107 of the mature protein and an NH2-terminal extension (presequence). The deduced amino acid sequence of the mature protein is identical with the previously determined protein sequence: the sequence of the NH2-terminal extension contains a potential initiator methionine at amino acid -22 from the NH2-terminus of the processed protein. The presequence is quite basic and contains several arginines, including one at the processing site. No hydrophobic region analogous to that found in bacterial and eukaryotic signal peptides is present, but there are homologies with other mitochondrial protein presequences, which may include a common signal for their destination and processing.

PMID: 6093095 [PubMed - indexed for MEDLINE]

A restriction enzyme cleavage map of the histidine utilization (hut) genes of Klebsiella aerogenes and deletions lacking regions of hut DNA.

Mol Gen Genet. 1984;193(1):92-8

Authors: Boylan SA, Eades LJ, Janssen KA, Lomax MI, Bender RA

Abstract
The histidine utilization (hut) operons of Klebsiella aerogenes were cloned into pBR322. The hut genes are wholly contained on a 7.9 kilobase pair fragment bounded by HindIII restriction sites and expression of hut is independent of the orientation of the fragment with respect to pBR322. A restriction map locating the 27 cleavage sites within hut for the enzymes, HindIII, PvuII, SalI, BglII, KpnI, PstI, SmaI, AvaI, and BamHI was deduced. Several of the cleavage sites for the enzymes HaeIII and HinfI were also mapped. A set of deletion plasmids was isolated by removing various restriction fragments from the original plasmid. These deletions were characterized and were used to assist in mapping restriction sites. This physical characterization of hut DNA opens the way for genetic and molecular analysis of the regulation of hut gene expression in vitro as well as in vivo.

PMID: 6318054 [PubMed - indexed for MEDLINE]

Cloned ribosomal RNA genes from chloroplasts of Euglena gracilis.

Science. 1977 Apr 8;196(4286):202-5

Authors: Lomax MI, Helling RB, Hecker LI, Schwartzbach SD, Barnett WE

Abstract
Fragments of Euglena chloroplast DNA generated by endonuclease R-Eco RI were separated by agarose-gel electrophoresis into 24 distinct bands. At least five fragments contain sequences complementary to chloroplast ribosomal RNA, Most of the Eco RI fragments have been cloned in a plasmid of Escherichia coli. Three of the cloned fragments were shown to contain chloroplast ribosomal RNA sequences by DNA-RNA hybridization.

PMID: 403604 [PubMed - indexed for MEDLINE]

An exchange between the hydrogen atom on carbon 5 of deoxyuridylate and water catalyzed by thymidylate synthetase.

J Biol Chem. 1967 Mar 25;242(6):1302-6

Authors: Lomax MI, Greenberg GR

PMID: 5337157 [PubMed - indexed for MEDLINE]

A new assay of thymidylate synthetase activity based on the release of tritium from deoxyuridylate-5-3-H.

J Biol Chem. 1967 Jan 10;242(1):109-13

Authors: Lomax MI, Greenberg GR

PMID: 5334158 [PubMed - indexed for MEDLINE]