Nicholas J Cowan, Ph.D.

Regulation of Androgen Receptor-Mediated Transcription by RPB5 Binding Protein URI/RMP
Mita, Paolo; Savas, Jeffrey N; Djouder, Nabil; Yates, John R 3rd; Ha, Susan; Ruoff, Rachel; Schafler, Eric D; Nwachukwu, Jerome C; Tanese, Naoko; Cowan, Nicholas J; Zavadil, Jiri; Garabedian, Michael J; Logan, Susan K
2011 Sep;31(17):3639-3652, Molecular & cellular biology
Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes
— id: 136514, year: 2011, vol: 31, page: 3639, stat: Journal Article,

Tuba8 Is Expressed at Low Levels in the Developing Mouse and Human Brain
Braun, A; Breuss, M; Salzer, MC; Flint, J; Cowan, NJ; Keays, DA
2010 MAY 14 ;86(5):819-822, American journal of human genetics
— id: 110005, year: 2010, vol: 86, page: 819, stat: Journal Article,

Disease-associated mutations in TUBA1A result in a spectrum of defects in the tubulin folding and heterodimer assembly pathway
Tian, Guoling; Jaglin, Xavier H; Keays, David A; Francis, Fiona; Chelly, Jamel; Cowan, Nicholas J
2010 Sep 15;19(18):3599-3613, Human molecular genetics
Malformations of cortical development are characteristic of a plethora of diseases that includes polymicrogyria, periventricular and subcortical heterotopia and lissencephaly. Mutations in TUBA1A and TUBB2B, each a member of the multigene families that encode alpha- and beta-tubulins, have recently been implicated in these diseases. Here we examine the defects that result from nine disease-causing mutations (I188L, I238V, P263T, L286F, V303G, L397P, R402C, 402H, S419L) in TUBA1A. We show that the expression of all the mutant proteins in vitro results in the generation of tubulin heterodimers in varying yield and that these can co-polymerize with microtubules in vitro. We identify several kinds of defects that result from these mutations. Among these are various defects in the chaperone-dependent pathway leading to de novo tubulin heterodimer formation. These include a defective interaction with the chaperone prefoldin, a reduced efficiency in the generation of productive folding intermediates as a result of inefficient interaction with the cytosolic chaperonin, CCT, and, in several cases, a failure to stably interact with TBCB, one of five tubulin-specific chaperones that act downstream of CCT in the tubulin heterodimer assembly pathway. Other defects include structural instability in vitro, diminished stability in vivo, a compromised ability to co-assemble with microtubules in vivo and a suppression of microtubule growth rate in the neurites (but not the soma) of cultured neurons. Our data are consistent with the notion that some mutations in TUBA1A result in tubulin deficit, whereas others reflect compromised interactions with one or more MAPs that are essential to proper neuronal migration
— id: 112037, year: 2010, vol: 19, page: 3599, stat: Journal Article,

Effect of TBCD and its regulatory interactor Arl2 on tubulin and microtubule integrity
Tian, Guoling; Thomas, Simi; Cowan, Nicholas J
2010 Nov;67(11):706-714, Cytoskeleton (Hoboken)
Assembly of the alpha/beta tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin (CCT) as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of alpha- and beta-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric cocomplex with beta-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with beta-tubulin. We show that recombinant human TBCD functions indistinguishably from its nonrecombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GTPase activating protein assays in which TBCD and TBCC stimulate GTP hydrolysis by beta-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP ribosylation factor-like protein). (c) 2010 Wiley-Liss, Inc
— id: 113945, year: 2010, vol: 67, page: 706, stat: Journal Article,

Mutations in the beta-tubulin gene TUBB2B result in asymmetrical polymicrogyria
Jaglin, Xavier Hubert; Poirier, Karine; Saillour, Yoann; Buhler, Emmanuelle; Tian, Guoling; Bahi-Buisson, Nadia; Fallet-Bianco, Catherine; Phan-Dinh-Tuy, Francoise; Kong, Xiang Peng; Bomont, Pascale; Castelnau-Ptakhine, Laetitia; Odent, Sylvie; Loget, Philippe; Kossorotoff, Manoelle; Snoeck, Irina; Plessis, Ghislaine; Parent, Philippe; Beldjord, Cherif; Cardoso, Carlos; Represa, Alfonso; Flint, Jonathan; Keays, David Anthony; Cowan, Nicholas Justin; Chelly, Jamel
2009 Jun;41(6):746-752, Nature genetics
Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. Here we report de novo mutations in a beta-tubulin gene, TUBB2B, in four individuals and a 27-gestational-week fetus with bilateral asymmetrical polymicrogyria. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to impaired formation of tubulin heterodimers. These observations, together with previous data, show that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly and pachygyria, but also polymicrogyria malformations
— id: 135247, year: 2009, vol: 41, page: 746, stat: Journal Article,

A Pachygyria-causing {alpha}-Tubulin Mutation Results in Inefficient Cycling with CCT and a Deficient Interaction with TBCB
Tian, Guoling; Kong, Xiang-Peng; Jaglin, Xavier H; Chelly, Jamel; Keays, David; Cowan, Nicholas J
2008 Mar;19(3):1152-1161, Molecular biology of the cell
The agyria (lissencephaly)/pachygyria phenotypes are catastrophic developmental diseases characterized by abnormal folds on the surface of the brain and disorganized cortical layering. In addition to mutations in at least four genes-LIS1, DCX, ARX and RELN-mutations in a human alpha-tubulin gene, TUBA1A, have recently been identified that cause these diseases. Here, we show that one such mutation, R264C, leads to a diminished capacity of de novo tubulin heterodimer formation. We identify the mechanisms that contribute to this defect. First, there is a reduced efficiency whereby quasinative alpha-tubulin folding intermediates are generated via ATP-dependent interaction with the cytosolic chaperonin CCT. Second, there is a failure of CCT-generated folding intermediates to stably interact with TBCB, one of the five tubulin chaperones (TBCA-E) that participate in the pathway leading to the de novo assembly of the tubulin heterodimer. We describe the behavior of the R264C mutation in terms of its effect on the structural integrity of alpha-tubulin and its interaction with TBCB. In spite of its compromised folding efficiency, R264C molecules that do productively assemble into heterodimers are capable of copolymerizing into dynamic microtubules in vivo. The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype
— id: 78375, year: 2008, vol: 19, page: 1152, stat: Journal Article,

Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans
Keays, David A; Tian, Guoling; Poirier, Karine; Huang, Guo-Jen; Siebold, Christian; Cleak, James; Oliver, Peter L; Fray, Martin; Harvey, Robert J; Molnar, Zoltan; Pinon, Maria C; Dear, Neil; Valdar, William; Brown, Steve D M; Davies, Kay E; Rawlins, J Nicholas P; Cowan, Nicholas J; Nolan, Patrick; Chelly, Jamel; Flint, Jonathan
2007 Jan 12;128(1):45-57, Cell
The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders
— id: 78376, year: 2007, vol: 128, page: 45, stat: Journal Article,

Cryptic out-of-frame translational initiation of TBCE rescues tubulin formation in compound heterozygous HRD
Tian, Guoling; Huang, Melissa C; Parvari, Ruti; Diaz, George A; Cowan, Nicholas J
2006 Sep 5;103(36):13491-13496, Proceedings of the National Academy of Sciences of the United States of America
Microtubules are indispensable dynamic structures that contribute to many essential biological functions. Assembly of the native alpha/beta tubulin heterodimer, the subunit that polymerizes to form microtubules, requires the participation of several molecular chaperones, namely prefoldin, the cytosolic chaperonin CCT, and a series of five tubulin-specific chaperones termed cofactors A-E (TBCA-E). Among these, TBCC, TBCD, and TBCE are essential in higher eukaryotes; they function together as a multimolecular machine that assembles quasinative CCT-generated alpha- and beta-tubulin polypeptides into new heterodimers. Deletion and truncation mutations in the gene encoding TBCE have been shown to cause the rare autosomal recessive syndrome known as HRD, a devastating disorder characterized by congenital hypoparathyroidism, mental retardation, facial dysmorphism, and extreme growth failure. Here we identify cryptic translational initiation at each of three out-of-frame AUG codons upstream of the genetic lesion as a unique mechanism that rescues a mutant HRD allele by producing a functional TBCE protein. Our data explain how afflicted individuals, who would otherwise lack the capacity to make functional TBCE, can survive and point to a limiting capacity to fold tubulin heterodimers de novo as a contributing factor to disease pathogenesis
— id: 67543, year: 2006, vol: 103, page: 13491, stat: Journal Article,

Mutations affecting beta-tubulin folding and degradation
Wang, Yaqing; Tian, Guoling; Cowan, Nicholas J; Cabral, Fernando
2006 May 12;281(19):13628-13635, Journal of biological chemistry
Revertants of a colcemid-resistant Chinese hamster ovary cell line with an altered (D45Y) beta-tubulin have allowed the identification of four cis-acting mutations (L187R, Y398C, a 12-amino acid in-frame deletion, and a C-terminal truncation) that act by destabilizing the mutant tubulin and preventing it from incorporating into microtubules. These unstable beta-tubulins fail to form heterodimers and are predominantly found in association with the chaperonin CCT, suggesting that they cannot undergo productive folding. In agreement with these in vivo observations, we show that the defective beta-tubulins do not stably interact with cofactors involved in the tubulin folding pathway and, hence, fail to exchange with beta-tubulin in purified alphabeta heterodimers. Treatment of cells with MG132 causes an accumulation of the aberrant tubulins, indicating that improperly folded beta-tubulin is degraded by the proteasome. Rapid degradation of the mutant tubulin does not elicit compensatory changes in wild-type tubulin synthesis or assembly. Instead, loss of beta-tubulin from the mutant allele causes a 30-40% decrease in cellular tubulin content with no obvious effect on cell growth or survival
— id: 67544, year: 2006, vol: 281, page: 13628, stat: Journal Article,

Identification of a novel tubulin-destabilizing protein related to the chaperone cofactor E
Bartolini, Francesca; Tian, Guoling; Piehl, Michelle; Cassimeris, Lynne; Lewis, Sally A; Cowan, Nicholas J
2005 Mar 15;118(Pt 6):1197-1207, Journal of cell science
Factors that regulate the microtubule cytoskeleton are critical in determining cell behavior. Here we describe the function of a novel protein that we term E-like based on its sequence similarity to the tubulin-specific chaperone cofactor E. We find that upon overexpression, E-like depolymerizes microtubules by committing tubulin to proteosomal degradation. Our data suggest that this function is direct and is based on the ability of E-like to disrupt the tubulin heterodimer in vitro. Suppression of E-like expression results in an increase in the number of stable microtubules and a tight clustering of endocellular membranes around the microtubule-organizing center, while the properties of dynamic microtubules are unaffected. These observations define E-like as a novel regulator of tubulin stability, and provide a link between tubulin turnover and vesicle transport
— id: 56011, year: 2005, vol: 118, page: 1197, stat: Journal Article,

Selective contribution of eukaryotic prefoldin subunits to actin and tubulin binding
Simons, C Torrey; Staes, An; Rommelaere, Heidi; Ampe, Christophe; Lewis, Sally A; Cowan, Nicholas J
2004 Feb 6;279(6):4196-4203, Journal of biological chemistry
Eukaryotic prefoldin (PFD) is a heterohexameric chaperone with a jellyfish-like structure whose function is to deliver nonnative target proteins, principally actins and tubulins, to the eukaryotic cytosolic chaperonin for facilitated folding. Here we demonstrate that functional PFD can spontaneously assemble from its six constituent individual subunits (PFD1-PFD6), each expressed as a recombinant protein. Using engineered forms of PFD assembled in vitro, we show that the tips of the PFD tentacles are required to form binary complexes with authentic target proteins. We show that PFD uses the distal ends of different but overlapping sets of subunits to form stable binary complexes with different target proteins, namely actin and alpha- and beta-tubulin. We also present data that suggest a model for the order of these six subunits within the hexamer. Our data are consistent with the hypothesis that PFD, like the eukaryotic cytosolic chaperonin, has co-evolved specifically to facilitate the folding of its target proteins
— id: 42643, year: 2004, vol: 279, page: 4196, stat: Journal Article,

Cytosolic Arl2 is complexed with cofactor D and protein phosphatase 2A
Shern, Jack F; Sharer, J Daniel; Pallas, David C; Bartolini, Francesca; Cowan, Nicholas J; Reed, Matthew S; Pohl, Jan; Kahn, Richard A
2003 Oct 17;278(42):40829-40836, Journal of biological chemistry
Arl2 is a member of the ADP-ribosylation factor family of 20-kDa GTPases that is highly conserved in eukaryotes. Recent results revealed that a portion of cellular Arl2 and its binding partner, BART, localize to mitochondria. Because approximately 90% of cellular Arl2 is cytosolic, we investigated properties of the soluble protein and found that it is stably bound in a complex that migrates in gel filtration medium with a predicted molecular mass of approximately 300 kDa. This complex was purified approximately 500-fold from the soluble fraction of bovine brain. Protein components were identified by mass spectroscopy and revealed the presence of four other proteins that include the tubulin folding cochaperone cofactor D and all three subunits of at least two protein phosphatase 2A (PP2A) protein phosphatase trimers. The presence of more than one PP2A B-type subunit and the low stoichiometry of Arl2 indicate that the purified preparation still contains a mixture of complexes that cannot currently be completely resolved. Thus, although all the soluble Arl2 in bovine brain is in high molecular mass complexes, only a portion of the total cellular cofactor D and PP2A are associated with the Arl2. We further show that the Arl2 in the complex cannot bind GTP and that complexed cofactor D does not efficiently participate in tubulin refolding reactions in a manner comparable with free cofactor D. Our data suggest functional roles for the cytosolic Arl2 complex in modulating tubulin and microtubule behavior as well as a possible role in apoptosis
— id: 48114, year: 2003, vol: 278, page: 40829, stat: Journal Article,

Functional overlap between retinitis pigmentosa 2 protein and the tubulin-specific chaperone cofactor C
Bartolini, Francesca; Bhamidipati, Arunashree; Thomas, Scott; Schwahn, Uwe; Lewis, Sally A; Cowan, Nicholas J
2002 Apr 26;277(17):14629-14634, Journal of biological chemistry
Mutations in the X-linked retinitis pigmentosa 2 gene cause progressive degeneration of photoreceptor cells. The retinitis pigmentosa 2 protein (RP2) is similar in sequence to the tubulin-specific chaperone cofactor C. Together with cofactors D and E, cofactor C stimulates the GTPase activity of native tubulin, a reaction regulated by ADP-ribosylation factor-like 2 protein. Here we show that in the presence of cofactor D, RP2 protein also stimulates the GTPase activity of tubulin. We find that this function is abolished by mutation in an arginine residue that is conserved in both cofactor C and RP2. Notably, mutations that alter this arginine codon cause familial retinitis pigmentosa. Our data imply that this residue acts as an 'arginine finger' to trigger the tubulin GTPase activity and suggest that loss of this function in RP2 contributes to retinal degeneration. We also show that in Saccharomyces cerevisiae, both cofactor C and RP2 partially complement the microtubule phenotype resulting from deletion of the cofactor C homolog, demonstrating their functional overlap in vivo. Finally, we find that RP2 interacts with GTP-bound ADP ribosylation factor-like 3 protein, providing a link between RP2 and several retinal-specific proteins, mutations in which also cause retinitis pigmentosa
— id: 67547, year: 2002, vol: 277, page: 14629, stat: Journal Article,

Localization in the human retina of the X-linked retinitis pigmentosa protein RP2, its homologue cofactor C and the RP2 interacting protein Arl3
Grayson, Celene; Bartolini, Francesca; Chapple, J Paul; Willison, Keith R; Bhamidipati, Arunashree; Lewis, Sally A; Luthert, Philip J; Hardcastle, Alison J; Cowan, Nicholas J; Cheetham, Michael E
2002 Nov 15;11(24):3065-3074, Human molecular genetics
Mutations in the retinitis pigmentosa 2 (RP2) gene cause a severe form of X-linked retinal degeneration. RP2 is a ubiquitous 350 amino acid plasma membrane-associated protein, which shares homology with the tubulin-specific chaperone cofactor C. RP2 protein, like cofactor C, stimulates the GTPase activity of tubulin in combination with cofactor D. RP2 has also been shown to interact with ADP ribosylation factor-like 3 (Arl3) in a nucleotide and myristoylation-dependant manner. In this study we have examined the relationship between RP2, cofactor C and Arl3 in patient-derived cell lines and in the retina. Examination of lymphoblastoid cells from patients with an Arg120stop nonsense mutation in RP2 revealed that the expression levels of cofactor C and Arl3 were not affected by the absence of RP2. In human retina, RP2 was localized to the plasma membrane of cells throughout the retina. RP2 was present at the plasma membrane in both rod and cone photoreceptors, extending from the outer segment through the inner segment to the synaptic terminals. There was no enrichment of RP2 staining in any photoreceptor organelle. In contrast, cofactor C and Arl3 localized predominantly to the photoreceptor connecting cilium in rod and cone photoreceptors. Cofactor C was cytoplasmic in distribution, whereas Arl3 localized to other microtubule structures within all cells. Arl3 behaved as a microtubule-associated protein: it co-localized with microtubules in HeLa cells and this was enhanced following microtubule stabilization with taxol. Furthermore, Arl3 co-purified with microtubules from bovine brain. Following microtubule depolymerization with nocodazole, Arl3 relocalized to the nuclear membrane. These data suggest that RP2 functions in concert with Arl3 to link the cell membrane with the cytoskeleton in photoreceptors as part of the cell signaling or vesicular transport machinery
— id: 67546, year: 2002, vol: 11, page: 3065, stat: Journal Article,

Bad chaperone
Lewis, Sally A; Cowan, Nicholas J
2002 Nov;8(11):1202-1203, Nature medicine
— id: 63336, year: 2002, vol: 8, page: 1202, stat: Journal Article,

Structure of eukaryotic prefoldin and of its complexes with unfolded actin and the cytosolic chaperonin CCT
Martin-Benito, Jaime; Boskovic, Jasminka; Gomez-Puertas, Paulino; Carrascosa, Jose L; Simons, C Torrey; Lewis, Sally A; Bartolini, Francesca; Cowan, Nicholas J; Valpuesta, Jose M
2002 Dec 2;21(23):6377-6386, EMBO journal
The biogenesis of the cytoskeletal proteins actin and tubulin involves interaction of nascent chains of each of the two proteins with the oligomeric protein prefoldin (PFD) and their subsequent transfer to the cytosolic chaperonin CCT (chaperonin containing TCP-1). Here we show by electron microscopy that eukaryotic PFD, which has a similar structure to its archaeal counterpart, interacts with unfolded actin along the tips of its projecting arms. In its PFD-bound state, actin seems to acquire a conformation similar to that adopted when it is bound to CCT. Three-dimensional reconstruction of the CCT:PFD complex based on cryoelectron microscopy reveals that PFD binds to each of the CCT rings in a unique conformation through two specific CCT subunits that are placed in a 1,4 arrangement. This defines the phasing of the CCT rings and suggests a handoff mechanism for PFD
— id: 67545, year: 2002, vol: 21, page: 6377, stat: Journal Article,

Type II chaperonins, prefoldin, and the tubulin-specific chaperones
Cowan NJ; Lewis SA
2001 ;59(2):73-104, Advances in protein chemistry
— id: 32463, year: 2001, vol: 59, page: 73, stat: Journal Article,

ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin
Bhamidipati A; Lewis SA; Cowan NJ
2000 May 29;149(5):1087-1096, Journal of cell biology
The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the alpha/beta- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a beta-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo
— id: 11674, year: 2000, vol: 149, page: 1087, stat: Journal Article,

The development of auditory attention in children
Gomes, H; Molholm, S; Christodoulou, C; Ritter, W; Cowan, N
2000 Jan 1;5:D108-D120, Frontiers in biosciences
In this paper we review the development of four components of auditory attention: arousal, orienting, selective attention and sustained attention. We focus especially on the processes responsible for the selection of specific stimuli for further processing because these are essential for learning and development. Although much work still needs to be done, there is evidence of developmental change in some of the components of attention, especially early in infancy. Later developmental improvements seem to be primarily attributable to higher cognitive processes, such as motivation, strategy development and implementation, and voluntary direction and regulation of attention
— id: 129227, year: 2000, vol: 5, page: D108, stat: Journal Article,

Mismatch negativity in children and adults, and effects of an attended task
Gomes, H; Molholm, S; Ritter, W; Kurtzberg, D; Cowan, N; Vaughan, H G Jr
2000 Nov;37(6):807-816, Psychophysiology
Attention has been shown to modulate the amplitude of the mismatch negativity (MMN) elicited by a small deviation in auditory stimuli in adults. The present study examined the effects of attention and deviant size on MMN amplitude in children. Children and adults were presented with sequences of tones containing standards (1000 Hz) and three deviants varying in degree of deviance from the standard (1050, 1200, and 1500 Hz). Tones were presented in three conditions: (1) while participants ignored them; (2) while participants listened to them and responded to all three deviants; and (3) while participants again ignored them. We found that the MMNs elicited by the hard deviant (1050 Hz) were larger when the children were actively discriminating the stimuli than when they were ignoring them. However, the MMNs elicited by the easy and medium deviants (1500 and 1200 Hz, respectively) in the children and by all three deviants in the adults were not affected by attention
— id: 129228, year: 2000, vol: 37, page: 807, stat: Journal Article,

Purification of prefoldin
Lewis, S A; Cowan, N J
2000 ;140:179-184, Methods in molecular biology
— id: 78378, year: 2000, vol: 140, page: 179, stat: Journal Article,

A chaperone with a hydrophilic surface
Cowan NJ; Lewis SA
1999 Nov;6(11):990-991, Nature structural biology
The folding of native tubulin involves at least seven different chaperone proteins: prefoldin, the cytosolic chaperonin CCT and five tubulin-specific chaperone proteins named cofactors A-E. The structure of the yeast homolog of cofactor A, Rbl2p, shows it to be a dimer with largely hydrophilic surfaces, reflecting the fact that it interacts with quasi-native, not unfolded, beta-tubulin
— id: 11910, year: 1999, vol: 6, page: 990, stat: Journal Article,

Prefoldin-nascent chain complexes in the folding of cytoskeletal proteins
Hansen WJ; Cowan NJ; Welch WJ
1999 Apr 19;145(2):265-277, Journal of cell biology
In vitro transcription/translation of actin cDNA and analysis of the translation products by native-PAGE was used to study the maturation pathway of actin. During the course of actin synthesis, several distinct actin-containing species were observed and the composition of each determined by immunological procedures. After synthesis of the first approximately 145 amino acids, the nascent ribosome-associated actin chain binds to the recently identified heteromeric chaperone protein, prefoldin (PFD). PFD remains bound to the relatively unfolded actin polypeptide until its posttranslational delivery to cytosolic chaperonin (CCT). We show that alpha- and beta-tubulin follow a similar maturation pathway, but to date find no evidence for an interaction between PFD and several noncytoskeletal proteins. We conclude that PFD functions by selectively targeting nascent actin and tubulin chains pending their transfer to CCT for final folding and/or assembly
— id: 17132, year: 1999, vol: 145, page: 265, stat: Journal Article,

A single-ring mitochondrial chaperonin (Hsp60-Hsp10) can substitute for GroEL-GroES in vivo
Nielsen KL; McLennan N; Masters M; Cowan NJ
1999 Sep;181(18):5871-5875, Journal of bacteriology
Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES
— id: 6201, year: 1999, vol: 181, page: 5871, stat: Journal Article,

Tubulin folding cofactors as GTPase-activating proteins. GTP hydrolysis and the assembly of the alpha/beta-tubulin heterodimer
Tian G; Bhamidipati A; Cowan NJ; Lewis SA
1999 Aug 20;274(34):24054-24058, Journal of biological chemistry
In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized alpha- and beta-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. alpha- and beta-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A-E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by beta-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein beta-tubulin to hydrolyze its GTP
— id: 6176, year: 1999, vol: 274, page: 24054, stat: Journal Article,

Mammalian cytosolic chaperonin
Cowan NJ
1998 ;290:230-241, Methods in enzymology
Cytosolic chaperonin, the eukaryotic cytosolic homolog of GroEL, has certain unusual features that make it uniquely useful for studying the mechanism of chaperonin action. It is of particular interest as an essential component in the generation of native actin and tubulin in vivo. We describe a method for the purification of mammalian c-cpn from rabbit reticulocyte lysate via a three-step procedure involving ion-exchange chromatography, affinity selection on ATP-agarose, and gel filtration. We also describe a sensitive in vitro-folding assay for the activity of c-cpn and other chaperone proteins, and a simple nondenaturing gel assay for the analysis of folding reaction products
— id: 12142, year: 1998, vol: 290, page: 230, stat: Journal Article,

Cytoskeletal protein-prefoldin chaperone complexes form co-translationally en route to the cytosolic chaperonin and folding to the native state
Hansen, WJ; Cowan, NJ; Welch, WJ
1998 NOV ;9(11):453A-453A, Molecular biology of the cell
— id: 53651, year: 1998, vol: 9, page: 453A, stat: Journal Article,

A single ring is sufficient for productive chaperonin-mediated folding in vivo
Nielsen KL; Cowan NJ
1998 Jul;2(1):93-99, Molecular cell
Facilitated protein folding by the double toroidal bacterial chaperonin, GroEL/GroES, proceeds by a 'two-stroke engine' mechanism in which an allosteric interaction between the two rings synchronizes the reaction cycle by controlling the binding and release of cochaperonin. Using chimeric chaperonin molecules assembled by fusing equatorial and apical domains derived from GroEL and its mammalian mitochondrial homolog, Hsp60, we show that productive folding by Hsp60 and its cognate cochaperonin, Hsp10, proceeds in vitro and in vivo without the formation of a two-ring structure. This simpler 'one-stroke' engine works because Hsp60 has a different mechanism for the release of its cochaperonin cap and bound target protein
— id: 7711, year: 1998, vol: 2, page: 93, stat: Journal Article,

Prefoldin, a chaperone that delivers unfolded proteins to cytosolic chaperonin
Vainberg IE; Lewis SA; Rommelaere H; Ampe C; Vandekerckhove J; Klein HL; Cowan NJ
1998 May 29;93(5):863-873, Cell
We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins
— id: 7835, year: 1998, vol: 93, page: 863, stat: Journal Article,

The alpha- and beta-tubulin folding pathways
Lewis, S A; Tian, G; Cowan, N J
1997 Dec;7(12):479-484, Trends in cell biology
The alpha-beta tubulin heterodimer is the subunit from which microtubules are assembled. The pathway leading to correctly folded alpha- and beta-tubulins is unusually complex: it involves cycles of ATP-dependent interaction of newly synthesized tubulin subunits with cytosolic chaperonin, resulting in the production of quasi-native folding intermediates, which must then be acted upon by additional protein cofactors. These cofactors form a supercomplex containing both alpha- and beta-tubulin polypeptides, from which native heterodimer is released in a GTP-dependent reaction. Here, we discuss the current state of our understanding of the function of cytosolic chaperonin and cofactors in tubulin folding
— id: 78377, year: 1997, vol: 7, page: 479, stat: Journal Article,

Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors
Tian G; Lewis SA; Feierbach B; Stearns T; Rommelaere H; Ampe C; Cowan NJ
1997 Aug 25;138(4):821-832, Journal of cell biology
The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other
— id: 7270, year: 1997, vol: 138, page: 821, stat: Journal Article,

Chaperonin-mediated folding of actin and tubulin
Lewis SA; Tian G; Vainberg IE; Cowan NJ
1996 Jan;132(1-2):1-4, Journal of cell biology
— id: 6929, year: 1996, vol: 132, page: 1, stat: Journal Article,

GFAP is necessary for the integrity of CNS white matter architecture and long-term maintenance of myelination
Liedtke W; Edelmann W; Bieri PL; Chiu FC; Cowan NJ; Kucherlapati R; Raine CS
1996 Oct;17(4):607-615, Neuron
To investigate the structural role of glial fibrillary acidic protein (GFAP) in vivo, mice carrying a null mutation in GFAP were generated. In 7/14 mutant animals older than 18 months of age, hydrocephalus associated with white matter loss was detected. Mutant mice displayed abnormal myelination including the presence of actively myelinating oligodendrocytes in adults, nonmyelinated axons in optic nerve, and reduced myelin thickness in spinal cord. White matter was poorly vascularized and the blood-brain barrier was structurally and functionally impaired. Astrocytic structure and function were abnormal, consisting of shortened astrocytic cell processes, decreased septation of white matter, and increased CNS extracellular space. Thus, GFAP expression is essential for normal white matter architecture and blood-brain barrier integrity, and its absence leads to late-onset CNS dysmyelination
— id: 17133, year: 1996, vol: 17, page: 607, stat: Journal Article,

Pathway leading to correctly folded beta-tubulin
Tian G; Huang Y; Rommelaere H; Vandekerckhove J; Ampe C; Cowan NJ
1996 Jul 26;86(2):287-296, Cell
We describe the complete beta-tubulin folding pathway. Folding intermediates produced via ATP-dependent interaction with cytosolic chaperonin undergo a sequence of interactions with four proteins (cofactors A, D, E, and C). The postchaperonin steps in the reaction cascade do not depend on ATP or GTP hydrolysis, although GTP plays a structural role in tubulin folding. Cofactors A and D function by capturing and stabilizing beta-tubulin in a quasi-native conformation. Cofactor E binds to the cofactor D-beta-tubulin complex; interaction with cofactor C then causes the release of beta-tubulin polypeptides that are committed to the native state. Sequence analysis identifies yeast homologs of cofactors D (cin1) and E (pac2), characterized by mutations that affect microtubule function
— id: 56896, year: 1996, vol: 86, page: 287, stat: Journal Article,

Quasi-native chaperonin-bound intermediates in facilitated protein folding
Tian G; Vainberg IE; Tap WD; Lewis SA; Cowan NJ
1995 Oct 13;270(41):23910-23913, Journal of biological chemistry
Chaperonins are known to facilitate protein folding, but their mechanism of action is not well understood. The fact that target proteins are released from and rebind to different chaperonin molecules ('cycling') during a folding reaction suggests that chaperonins function by unfolding aberrantly folded molecules, allowing them multiple opportunities to reach the native state in bulk solution. Here we show that the cycling of alpha-tubulin by cytosolic chaperonin (c-cpn) can be uncoupled from the action of cofactors required to complete the folding reaction. This results in the accumulation of folding intermediates which are chaperonin-bound, stable, and quasi-native in that they bind GTP nonexchangeably. We present evidence that these intermediates can be generated without the target protein leaving c-cpn. These data show that, in contrast to prevailing models, target proteins can maintain, and possibly acquire, significant native-like structure while chaperonin-bound
— id: 6872, year: 1995, vol: 270, page: 23910, stat: Journal Article,

Specificity in chaperonin-mediated protein folding
Tian G; Vainberg IE; Tap WD; Lewis SA; Cowan NJ
1995 May 18;375(6528):250-253, Nature
Chaperonins are ubiquitous multisubunit toroidal complexes that aid protein folding in an ATP-dependent manner. Current models of folding by the bacterial chaperonin GroEL depict its role as unfolding and releasing molecules that have misfolded, so that they can return to a potentially productive folding pathway in solution. Accordingly, a given target polypeptide might require several cycles of binding and ATP-driven release from different chaperonin complexes before reaching the native state. Surprisingly, cycling of a target protein does not guarantee its folding, and we report here that unfolded beta-actin or alpha-tubulin both form tight complexes when presented to either GroEL or its mitochondrial homologue, and both undergo cycles of release and rebinding upon incubation with ATP, but no native protein is produced. We conclude that different chaperonins produce distinctive spectra of folding intermediates
— id: 56720, year: 1995, vol: 375, page: 250, stat: Journal Article,

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin
Gao Y; Melki R; Walden PD; Lewis SA; Ampe C; Rommelaere H; Vandekerckhove J; Cowan NJ
1994 Jun;125(5):989-996, Journal of cell biology
The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES
— id: 6381, year: 1994, vol: 125, page: 989, stat: Journal Article,

Facilitated folding of actins and tubulins occurs via a nucleotide-dependent interaction between cytoplasmic chaperonin and distinctive folding intermediates
Melki R; Cowan NJ
1994 May;14(5):2895-2904, Molecular & cellular biology
In the cytoplasm of eukaryotes, the folding of actins and tubulins is facilitated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin). The folding reaction consists of the formation of a binary complex between the unfolded target protein and the chaperonin, followed by the ultimate release of the native polypeptide in an ATP-dependent reaction. Here we show that the mitochondrial chaperonin (cpn60) and the cytoplasmic chaperonin both recognize a range of target proteins with different relative affinities; however, the cytoplasmic chaperonin shows the highest affinity for intermediates derived from unfolded tubulins and actins. These high-affinity actin and tubulin folding intermediates are distinct from the 'molten globule' intermediates formed by noncytoskeletal target proteins in that they form relatively slowly. We show that the interaction between cytoplasmic chaperonin and unfolded target proteins depends on the chaperonin being in its ADP-bound state and that the release of the target protein occurs after a transition of the chaperonin to the ATP-bound state. Our data suggest a model in which ATP hydrolysis acts as a switch between conformational forms of the cytoplasmic chaperonin that interact either strongly or weakly with unfolded substrates
— id: 17134, year: 1994, vol: 14, page: 2895, stat: Journal Article,

THE DEVELOPMENT OF CATEGORIES AT THE SEMANTICS SYNTAX INTERFACE
BRAINE, MDS; BROOKS, PJ; COWAN, N; SAMUELS, MC; TAMISLEMONDA, C
1993 OCT-DEC ;8(4):465-494, Cognitive development
This work aimed to reveal the thematic and grammatical role categories that are accessible to preschool children, and to determine how access to these categories changes with age. Three experiments explored categories associated with subjects of predicate adjectives and intransitive verbs, and with arguments of actional and experiential transitive verbs. A metalinguistic task was used in which children placed tokens on the objects in a picture according to the role they played in a sentence describing the scene. The results provide evidence for the psychological reality of a number of categorical distinctions. They also show greater salience of semantic categories relative to grammatical categories in young children, as compared with older children and adults, and they provide evidence bearing on theories of the development basis of subject. In particular, they are hard to reconcile with current versions of either Pinker's (1984) bootstrapping or Schlesinger's (1982, 1988) semantic assimilation theories, and a third theory is proposed
— id: 98446, year: 1993, vol: 8, page: 465, stat: Journal Article,

Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin
Gao Y; Vainberg IE; Chow RL; Cowan NJ
1993 Apr;13(4):2478-2485, Molecular & cellular biology
Though the chaperonins that mediate folding in prokaryotes, mitochondria, and chloroplasts have been relatively well characterized, the folding of proteins in the eukaryotic cytosol is much less well understood. We recently identified a cytoplasmic chaperonin as an 800-kDa multisubunit toroid which forms a binary complex with unfolded actin; the correctly folded polypeptide is released upon incubation with Mg-ATP (Y. Gao, J. O. Thomas, R. L. Chow, G.-H. Lee, and N. J. Cowan, Cell 69:1043-1050, 1992). Here we show that the same chaperonin also forms a binary complex with unfolded alpha- or beta-tubulin; however, there is no detectable release of the correctly folded product, irrespective of the concentration of added Mg-ATP and Mg-GTP or the presence of added carrier tubulin heterodimers with which newly folded alpha- or beta-tubulin polypeptides might exchange. Rather, two additional protein cofactors are required for the generation of properly folded alpha- or beta-tubulin, which is then competent for exchange into preexisting alpha/beta-tubulin heterodimers. We show that actin and tubulins compete efficiently with one another for association with cytoplasmic chaperonin complexes. These data imply that actin and alpha- and beta-tubulin interact with the same site(s) on chaperonin complexes
— id: 13211, year: 1993, vol: 13, page: 2478, stat: Journal Article,

Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin
Melki R; Vainberg IE; Chow RL; Cowan NJ
1993 Sep;122(6):1301-1310, Journal of cell biology
The folding of actin and tubulin is mediated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin) that hydrolyzes ATP as part of the reaction whereby native proteins are ultimately released. Vertebrate actin-related protein (actin-RPV) (also termed centractin) and gamma-tubulin are two proteins that are distantly related to actin and tubulin, respectively: gamma-tubulin is exclusively located at the centrosome, while actin-RPV is conspicuously abundant at the same site. Here we show that actin-RPV and gamma-tubulin are both folded via interaction with the same chaperonin that mediates the folding of beta-actin and alpha- and beta-tubulin. In each case, the unfolded polypeptide forms a binary complex with cytoplasmic chaperonin and is released as a soluble, monomeric protein in the presence of Mg-ATP and the presence or absence of Mg-GTP. In contrast to alpha- and beta-tubulin, the folding of gamma-tubulin does not require the presence of cofactors in addition to chaperonin itself. Monomeric actin-RPV produced in in vitro folding reactions cocycles efficiently with native brain actin, while in vitro folded gamma-tubulin binds to polymerized microtubules in a manner consistent with interaction with microtubule ends. Both monomeric actin-RPV and gamma-tubulin bind to columns of immobilized nucleotide: monomeric actin-RPV has no marked preference for ATP or GTP, while gamma-tubulin shows some preference for GTP binding. We show that actin-RPV and gamma-tubulin compete with one another, and with beta-actin or alpha-tubulin, for binary complex formation with cytoplasmic chaperonin
— id: 6454, year: 1993, vol: 122, page: 1301, stat: Journal Article,

Eukaryotic cytosolic chaperonin contains t-complex polypeptide 1 and seven related subunits
Rommelaere H; Van Troys M; Gao Y; Melki R; Cowan NJ; Vandekerckhove J; Ampe C
1993 Dec 15;90(24):11975-11979, Proceedings of the National Academy of Sciences of the United States of America
We have characterized the cytosolic chaperonin from both rabbit reticulocyte lysate and bovine testis. The heteromeric complex contains eight subunits. Partial amino acid sequence data reveal that one of these is t-complex polypeptide 1 (TCP-1), while the other seven are TCP-1-related polypeptides, implicating the existence of a multigene family of TCP-1 homologues. We provide evidence that TCP-1 ring complex from bovine testis can facilitate the folding of both actin and tubulin, although, as in the case of chaperonin from reticulocyte lysate, two cofactors are required for the generation of properly folded tubulin. An additional molecule of TCP-1 may associate with the chaperonin depending on the purification procedure used. We propose that a highly conserved region in these polypeptides and in other chaperonins of the cpn60 chaperone family participates in ATP binding
— id: 17135, year: 1993, vol: 90, page: 11975, stat: Journal Article,

A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette
Walden PD; Cowan NJ
1993 Dec;13(12):7625-7635, Molecular & cellular biology
To identify proteins which interact with and potentially modulate the function of microtubules during spermatogenesis, we prepared a total testis MAP (microtubule-associated protein) antiserum and used it to isolate cDNA clones from a mouse testis cDNA expression library. Antibodies affinity purified by using one expression clone recognized a 205-kDa protein, termed MAST205, which colocalizes with the spermatid manchette. Sequencing of full-length cDNA clones encoding MAST205 revealed it to be a novel serine/threonine kinase with a catalytic domain related to those of the A and C families. The testis-specific MAST205 RNA increases in abundance during prepuberal testis development, peaking at the spermatid stage. The microtubule-binding region of MAST205 occupies a central region of the molecule including the kinase domain and sequences C terminal to this domain. Binding of MAST205 to microtubules requires interaction with other MAPs, since it does not bind to MAP-free tubulin. A 75-kDa protein associated with immunoprecipitates of MAST205 from extracts of both whole testis and testis microtubules becomes phosphorylated in in vitro kinase assays. This 75-kDa substrate of the MAST205 kinase may form part of the MAST205 protein complex which binds microtubules. The MAST205 protein complex may function to link the signal transduction pathway with the organization of manchette microtubules
— id: 6548, year: 1993, vol: 13, page: 7625, stat: Journal Article,

The mechanism of equilibrium binding of microtubule-associated protein 2 to microtubules. Binding is a multi-phasic process and exhibits positive cooperativity
Wallis KT; Azhar S; Rho MB; Lewis SA; Cowan NJ; Murphy DB
1993 Jul 15;268(20):15158-15167, Journal of biological chemistry
The mechanism of binding of microtubule-associated protein 2 (MAP2) to taxol-stabilized microtubules (MTs) was examined through Scatchard analysis of equilibrium binding and by immunoelectron microscopy. We demonstrate the following. 1) Binding is a cooperative process as indicated by sigmoidal binding curves, prominent humps in Scatchard plots, and an all-or-none response in binding during ligand titrations. At high tubulin/MAP2 ratios, the Kd for noncontiguous binding (5-25 microM) is estimated to be 100-1500 times greater than that predicted for contiguous binding, suggesting a high degree of cooperativity. 2) Cooperativity is indicated independently by a highly clustered or patchy distribution of MAP2 on MTs as revealed by immunoelectron microscopy. 3) The binding of truncated constructs of mouse MAP2 protein suggests that a domain of MAP2 conferring cooperativity is located in or near the MT binding site near the carboxyl terminus. We speculate that in the cell, cooperativity may generate MTs with uniform biochemical properties and contribute to the segregation of MAPs in neuronal cell processes
— id: 17136, year: 1993, vol: 268, page: 15158, stat: Journal Article,

Projection domains of MAP2 and tau determine spacings between microtubules in dendrites and axons
Chen J; Kanai Y; Cowan NJ; Hirokawa N
1992 Dec 17;360(6405):674-677, Nature
Neurons develop a highly polarized morphology consisting of dendrites and a long axon. Both axons and dendrites contain microtubules and microtubule-associated proteins (MAPs) with characteristic structures. Among MAPs, MAP2 is specifically expressed in dendrites whereas MAP2C and tau are abundant in the axon. But the influence of MAP2, MAP2C and tau on the organization of microtubule domains in dendrites versus axons is unknown. Both MAP2 and tau induce microtubule bundle formation in fibroblasts after transfection of complementary DNAs, and a long process resembling an axon is extended in Sf9 cells infected with recombinant baculovirus expressing tau. We have now expressed MAP2 and MAP2C in Sf9 cells in order to compare their morphology and the arrangement of their microtubules to that found in Sf9 cells expressing tau. We report here that the spacing between microtubules depends on the MAP expressed: in cells expressing MAP2, the distance is similar to that found in dendrites, whereas the spacing between microtubules in cells expressing MAP2C or tau is similar to that found in axons
— id: 17137, year: 1992, vol: 360, page: 674, stat: Journal Article,

DIFFERENTIAL FUNCTIONS OF MAP2, MAP2C AND TAU ON THE ORGANIZATION OF MICROTUBULES IN THE CELLS
CHEN, J; KANAI, Y; COWAN, N; HIROKAWA, N
1992 SEP ;3(4):A259-A259, Molecular biology of the cell
— id: 51865, year: 1992, vol: 3, page: A259, stat: Journal Article,

A cytoplasmic chaperonin that catalyzes beta-actin folding
Gao Y; Thomas JO; Chow RL; Lee GH; Cowan NJ
1992 Jun 12;69(6):1043-1050, Cell
We have isolated a cytoplasmic chaperonin based on its ability to catalyze the folding of denatured beta-actin. The cytoplasmic chaperonin is organized as a multisubunit toroid and requires Mg2+ and ATP for activity. The folding reaction proceeds via the rapid ATP-independent formation of a binary complex, followed by a slower ATP-dependent release of the native product. Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg2+ and ATP. The eukaryotic cytoplasm thus contains a chaperonin that is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts
— id: 13562, year: 1992, vol: 69, page: 1043, stat: Journal Article,

Increased microtubule stability and alpha tubulin acetylation in cells transfected with microtubule-associated proteins MAP1B, MAP2 or tau
Takemura R; Okabe S; Umeyama T; Kanai Y; Cowan NJ; Hirokawa N
1992 Dec;103(Pt 4):953-964, Journal of cell science
We previously transfected MAP2, tau and MAP1B cDNA into fibroblasts and have studied the effect of expression of these microtubule-associated proteins on microtubule organization. In this study, we examined some additional characteristics of microtubule bundles and arrays formed in fibroblasts transfected with these microtubule-associated proteins. It was found that microtubule bundles formed in MAP2c- or tau-transfected cells were stabilized against microtubule depolymerizing reagents and were enriched in acetylated alpha tubulin. When mouse MAP1B cDNA was expressed following transfection into COS cells, MAP1B was localized along microtubule arrays, but no extensive reorganization of microtubules such as bundle formation was observed, in agreement with our previous finding using HeLa and 3T3 cells. However, stabilization of microtubules was indicated: (a) microtubules in MAP1B-transfected cells were stabilized against a microtubule depolymerizing reagent, although stabilization was less efficient than that seen in MAP2c- or tau-transfected cells, and (b) microtubules in MAP1B-transfected cells were enriched in acetylated alpha tubulin. These results suggest that neuronal microtubule-associated proteins introduced into fibroblasts by cDNA transfection stabilize microtubules and affect the state of post-translational modification of tubulin
— id: 17138, year: 1992, vol: 103, page: 953, stat: Journal Article,

Mammalian mitochondrial chaperonin 60 functions as a single toroidal ring
Viitanen PV; Lorimer GH; Seetharam R; Gupta RS; Oppenheim J; Thomas JO; Cowan NJ
1992 Jan 15;267(2):695-698, Journal of biological chemistry
Chaperonins are thought to participate in the process of protein folding in bacteria and in eukaryotic mitochondria and chloroplasts. While some chaperonins are relatively well characterized, the structures of the mammalian chaperonins are unknown. We have expressed a mammalian mitochondrial chaperonin 60 in Escherichia coli and purified the recombinant protein to homogeneity. Structural and biochemical analyses of this protein establish a single toroidal structure of seven subunits, in contrast to the homologous bacterial, fungal, and plant chaperonin 60s, which have double toroidal structures comprising two layers of seven identical subjects each. The recombinant mammalian chaperonin 60, together with the mammalian chaperonin 10 (but not with bacterial chaperonin 10), facilitates the formation of catalytically active ribulose-bisphosphate carboxylase from an unfolded state in the presence of K+ and MgATP. Analysis of the partial reactions involved in this in vitro reconstitution reveals that the single toroid of chaperonin 60 can form stable complexes with both unfolded or partially folded [35S]ribulose-bisphosphate carboxylase and mitochondrial (but not bacterial) chaperonin 10 in the presence of MgATP. We conclude that the minimal functional unit of chaperonin 60 is a single hepatmeric toroid
— id: 8274, year: 1992, vol: 267, page: 695, stat: Journal Article,

Tubulin dimer formation via the release of alpha- and beta-tubulin monomers from multimolecular complexes
Zabala JC; Cowan NJ
1992 ;23(3):222-230, Cell motility & the cytoskeleton
The functional subunit of microtubules is a heterodimer consisting of alpha- and beta-tubulin. An understanding of tubulin dimerization has been hampered because it has not proved possible to purify native tubulin monomers. To study the process whereby tubulin dimers are formed, we made use of tubulins synthesized by in vitro transcription and translation. We present evidence that the in vitro synthesis of different mouse alpha-tubulin isotypes involves a multimolecular complex. The synthesis of mouse beta-tubulin isotypes also involves the formation of multimolecular complexes, though different isotypes behave somewhat differently from one another. The properties of in vitro synthesized alpha- and beta-tubulin multimolecular complexes strongly suggest that they are intermediates in the biosynthesis of tubulin monomers. Upon release, these monomers can exchange with pre-existing tubulin heterodimers
— id: 17139, year: 1992, vol: 23, page: 222, stat: Journal Article,

THE NEURONAL CYTOSKELETON - BURGOYNE,RO
COWAN, N
1991 AUG 15 ;352(6336):580-580, Nature
— id: 51568, year: 1991, vol: 352, page: 580, stat: Journal Article,

Regulation of expression of glial filament acidic protein
Sarkar S; Cowan NJ
1991 ;15:97-102, Journal of cell science. Supplement
The regulation of cell type-specific expression of the gene encoding glial filament acidic protein (GFAP) was examined by introducing various deletion mutants of the gene into GFAP-expressing (U251 human astrocytoma) and non-expressing (HeLa) cell lines, and measuring their transcriptional activity in an RNAase protection assay. The expression of GFAP is influenced by a number of cis-acting elements. A domain that resides between nucleotides -1631 and -1479 can confer cell type-specific expression when coupled to a heterologous gene. We also present evidence for the existence of a negative regulatory element that resides within the first intron of the GFAP gene
— id: 14205, year: 1991, vol: 15, page: 97, stat: Journal Article,

INTRAGENIC SEQUENCES AFFECT THE EXPRESSION OF THE GENE ENCODING GLIAL FIBRILLARY ACIDIC PROTEIN
Sarkar, S; Cowan, NJ
1991 Aug;57(2):675-684, Journal of neurochemistry
We show that the expression of the gene encoding glial fibrillary acidic protein (GFAP) gene is affected by at least three cis-acting elements. A positive regulatory element that is located between nucleotides -1,631 and -1,479 can confer cell type-specific expression on a heterologous gene. A second regulatory element is located between nucleotides -97 and -80. The third is a negative regulatory element that is located within the first intron of the gene. Deletion of this element activates GFAP expression in HeLa cells, and affects promoter function in glioma cells
— id: 32165, year: 1991, vol: 57, page: 675, stat: Journal Article,

MICROTUBULE BUNDLING
Lewis, SA; Cowan, N
1990 Jun 21;345(6277):674-674, Nature
— id: 31929, year: 1990, vol: 345, page: 674, stat: Journal Article,

Assembly properties of altered beta-tubulin polypeptides containing disrupted autoregulatory domains
Gu W; Cowan NJ
1989 Aug;9(8):3418-3428, Molecular & cellular biology
beta-Tubulin synthesis in eucaryotic cells is subject to control by an autoregulatory posttranscriptional mechanism in which the first four amino acids of the beta-tubulin polypeptide act either directly or indirectly to control the stability of beta-tubulin mRNA. To investigate the contribution of this amino-terminal domain to microtubule assembly and dynamics, we introduced a series of deletions encompassing amino acids 2 to 5 of a single mammalian beta-tubulin isotype, M beta 1. Constructs carrying such deletions were inserted into an expression vector, and the ability of the altered polypeptide to coassemble into microtubules was tested by using an anti-M beta 1-specific antibody. We show that the M beta 1 beta-tubulin polypeptide was competent for coassembly into microtubules in transient transfection experiments and in stably transfected cell lines when it lacked either amino acid 2 or amino acids 2 and 3. The capacity of these mutant beta-tubulins to coassemble into polymerized microtubules was only slightly diminished relative to that of unaltered beta-tubulin, and their expression did not influence the viability or growth properties of cell lines carrying these deletions. However, more extensive amino-terminal deletions either severely compromised or abolished the capacity for coassembly. In analogous experiments in which alterations were introduced into the amino-terminal domain of a mammalian alpha-tubulin isotype, M alpha 4, deletion of amino acid 2 did not affect the ability of the altered polypeptide to coassemble, although removal of additional amino-terminal residues essentially abolished the capacity for competent coassembly. The stability of the altered assembly-competent alpha- and beta-tubulin polypeptides was measured in pulse-chase experiments and found to be indistinguishable from the stability of the corresponding unaltered polypeptides. An assembly-competent M alpha 4 polypeptide carrying a deletion encompassing the 12 carboxy-terminal amino acids also had a half-life indistinguishable from that of the wild-type alpha-tubulin molecule. These data suggest that the universally conserved amino terminus of beta-tubulin acts largely in a regulatory role and that the carboxy-terminal domain of alpha-tubulin is not essential for coassembly in mammalian cells in vivo
— id: 10532, year: 1989, vol: 9, page: 3418, stat: Journal Article,

Organization of microtubules in dendrites and axons is determined by a short hydrophobic zipper in microtubule-associated proteins MAP2 and tau
Lewis SA; Ivanov IE; Lee GH; Cowan NJ
1989 Nov 30;342(6249):498-505, Nature
Here we report that the microtubule-associated proteins MAP2 and tau share two separable functional domains. One is the microtubule-binding site which serves to nucleate microtubule assembly; the second is a short C-terminal alpha-helical sequence which can crosslink microtubules by means of a hydrophobic zipper interaction into dense stable parallel arrays characteristic of axons or dendrites. Thus, interactions between molecules of a single type are capable of drastically reorganizing microtubules and completely suppressing their dynamic properties
— id: 10427, year: 1989, vol: 342, page: 498, stat: Journal Article,

The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau
Noble M; Lewis SA; Cowan NJ
1989 Dec;109(6 Pt 2):3367-3376, Journal of cell biology
We report the complete sequence of the microtubule-associated protein MAP1B, deduced from a series of overlapping genomic and cDNA clones. The encoded protein has a predicted molecular mass of 255,534 D and contains two unusual sequences. The first is a highly basic region that includes multiple copies of a short motif of the form KKEE or KKEVI that are repeated, but not at exact intervals. The second is a set of 12 imperfect repeats, each of 15 amino acids and each spaced by two amino acids. Subcloned fragments spanning these two distinctive regions were expressed as labeled polypeptides by translation in a cell-free system in vitro. These polypeptides were tested for their ability to copurify with unlabeled brain microtubules through successive cycles of polymerization and depolymerization. The peptide corresponding to the region containing the KKEE and KKEVI motifs cycled with brain microtubules, whereas the peptide corresponding to the set of 12 imperfect repeats did not. To define the microtubule binding domain in vivo, full-length and deletion constructs encoding MAP1B were assembled and introduced into cultured cells by transfection. The expression of transfected polypeptides was monitored by indirect immunofluorescence using anti-MAP1B-specific antisera. These experiments showed that the basic region containing the KKEE and KKEVI motifs is responsible for the interaction between MAP1B and microtubules in vivo. This region bears no sequence relationship to the microtubule binding domains of kinesin, MAP2, or tau
— id: 10424, year: 1989, vol: 109, page: 3367, stat: Journal Article,

Nerve growth factor regulates both the phosphorylation and steady-state levels of microtubule-associated protein 1.2 (MAP1.2)
Aletta JM; Lewis SA; Cowan NJ; Greene LA
1988 May;106(5):1573-1581, Journal of cell biology
This study characterizes effects of nerve growth factor (NGF) on the steady-state level and phosphorylation of a high molecular mass microtubule-associated protein in PC12 rat pheochromocytoma cells. Past work showed that NGF significantly raises the relative levels of this phosphoprotein, designated MAP1.2, with a time course similar to that of neurite outgrowth. To study this in greater detail, MAP1.2 in PC12 cell lysates was resolved by SDS-PAGE in gels containing 3.25% acrylamide/4 M urea and identified by comigration with material immunoprecipitated from the lysates by MAP1 antibodies. Quantification by metabolic radiolabeling with [35S]methionine or by silver staining revealed a 3.0-3.5-fold increase in MAP1.2 levels relative to total cell protein after NGF treatment for 2 wk or longer. A partial increase was detectable after 3 d, but not after 2 h of NGF exposure. As measured by incorporation of [32P]phosphate, NGF had a dual effect on MAP1.2. Within 15 min to 2 h, NGF enhanced the incorporation of phosphate into MAP1.2 by two- to threefold relative to total cell phosphoproteins. This value slowly increased thereafter so that by 2 wk or more of NGF exposure, the average enhancement of phosphate incorporation per MAP1.2 molecule was over fourfold. The rapid action of NGF on MAP1.2 could not be mimicked by either epidermal growth factor, a permeant cAMP derivative, phorbol ester, or elevated K+, each of which alters phosphorylation of other PC12 cell proteins. SDS-PAGE revealed multiple forms of MAP1.2 which, based on the effects of alkaline phosphatase on their electrophoretic mobilities, differ, at least in part, in extent of phosphorylation. Before NGF treatment, most PC12 cell MAP1.2 is in more rapidly migrating, relatively poorly phosphorylated forms. After long-term NGF exposure, most is in more slowly migrating, more highly phosphorylated forms. The effects of NGF on the rapid phosphorylation of MAP1.2 and on the long-term large increase in highly phosphorylated MAP1.2 forms could play major functional roles in NGF-mediated neuronal differentiation. Such roles may include effects on microtubule assembly, stability, and cross-linking and, possibly for the rapid effects, nuclear signaling
— id: 11102, year: 1988, vol: 106, page: 1573, stat: Journal Article,

Differential distribution of beta-tubulin isotypes in cerebellum
Burgoyne RD; Cambray-Deakin MA; Lewis SA; Sarkar S; Cowan NJ
1988 Aug;7(8):2311-2319, EMBO journal
We describe the structure and expression of a mammalian beta-tubulin isotype (M beta 6) that is weakly expressed in testis but is abundant in developing brain, with transcripts declining to lower levels in the adult brain. The expression of M beta 6 was undetectable in any other mouse tissue examined. A serum specific for this isotype was prepared using a cloned fusion protein as immunogen. M beta 6 is one of five known beta-tubulin isotypes expressed in brain, and using the anti-M beta 6 serum along with sera, anti-M beta 2, anti-M beta 3/4 and anti-M beta 5, previously characterized, we have examined the pattern of expression of beta-tubulin isotypes in rat cerebellum. The isotypes each have characteristic cell-type specific patterns of localization in cerebellum. M beta 2, M beta 3/4 and M beta 5 are present in both neuronal and non-neuronal cells, but in contrast M beta 6 was only detectable in neurons in tissue sections and in dissociated cerebellar cell culture. The majority of sequence differences among the beta-tubulin isotypes lie at the carboxy terminus, the region of beta-tubulin involved in MAP binding. In the case of M beta 2 and M beta 6, the patterns of expression are similar or identical to the patterns of expression of MAP3 and MAP1A respectively. These results suggest that beta-tubulin isotypes may contribute to the determination of the specific association of MAPs with microtubules of diverse function. However, the strict subcellular segregation of other MAPs in brain may be determined by other factors
— id: 17140, year: 1988, vol: 7, page: 2311, stat: Journal Article,

TUBULIN ISOTYPES AND THEIR INTERACTION WITH MICROTUBULE ASSOCIATED PROTEINS
Cowan, NJ; Lewis, SA; Wei, G; Burgoyne, RD
1988 Sep;145(2-3):106-111, Protoplasma
— id: 31591, year: 1988, vol: 145, page: 106, stat: Journal Article,

Generation of antisera that discriminate among mammalian alpha-tubulins: introduction of specialized isotypes into cultured cells results in their coassembly without disruption of normal microtubule function
Gu W; Lewis SA; Cowan NJ
1988 Jun;106(6):2011-2022, Journal of cell biology
To assay the functional significance of the multiple but closely related alpha-tubulin polypeptides that are expressed in mammalian cells, we generated three specific immune sera, each of which uniquely recognizes a distinct alpha-tubulin isotype. All three isotypes are expressed in a tissue-restricted manner: one (M alpha 3/7) only in mature testis, one (M alpha 4) mainly in muscle and brain, and the third (M alpha 6) in several tissues at a very low level. A fourth specific antiserum was also generated that distinguishes between the tyrosinated and nontyrosinated form of a single alpha-tubulin isotype. Because individual tubulin isotypes cannot be purified biochemically, these sera were raised using cloned fusion proteins purified from host Escherichia coli cells. To suppress the immune response to shared epitopes, animals were first rendered tolerant to fusion proteins encoding all but one of the known mammalian alpha-tubulin isotypes. Subsequent challenge with the remaining fusion protein then resulted in the elicitation of an immune response to unique epitopes. Three criteria were used to establish the specificity of the resulting sera: (a) their ability to discriminate among cloned fusion proteins representing all the known mammalian alpha-tubulin isotypes; (b) their ability to uniquely detect alpha-tubulin in whole extracts of tissues; and (c) their capacity to stain microtubules in fixed preparations of cells transfected with sequences encoding the corresponding isotype. The transfection experiments served to demonstrate (a) the coassembly of M alpha 3/7, M alpha 4, and M alpha 6 into both interphase and spindle microtubules in HeLa cells and NIH 3T3 cells, and (b) that the M alpha 4 isotype, which is unique among mammalian alpha-tubulins in that it lacks an encoded carboxy-terminal tyrosine residue, behaves like other alpha-tubulin isotypes with respect to the cycle of tyrosination/detyrosination that occurs in most cultured cells
— id: 11078, year: 1988, vol: 106, page: 2011, stat: Journal Article,

THE PRIMARY STRUCTURE AND HETEROGENEITY OF TAU-PROTEIN FROM MOUSE-BRAIN
LEE, G; COWAN, N; KIRSCHNER, M
1988 JAN 15 ;239(4837):285-288, Science
— id: 41864, year: 1988, vol: 239, page: 285, stat: Journal Article,

Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells
Lewis SA; Cowan NJ
1988 Jun;106(6):2023-2033, Journal of cell biology
In the accompanying paper (Gu, W., S. A. Lewis, and N. J. Cowan. 1988. J. Cell Biol. 106: 2011-2022), we report the generation of three antisera, each of which uniquely recognizes a different mammalian alpha-tubulin isotype, plus a fourth antibody that distinguishes between microtubules containing the tyrosinated and nontyrosinated form of the only known mammalian alpha-tubulin gene product that lacks an encoded carboxy-terminal tyrosine residue. These sera, together with five sera we raised that distinguish among the known mammalian beta-tubulin isotypes, have been used to study patterns of tubulin isotype-specific expression in muscle and testis, two tissues in which characteristic developmental changes are accompanied by dramatic rearrangements in microtubule structures. As in the case of cells in culture, there is no evidence to suggest that there is subcellular sorting of different tubulin isotypes among different kinds of microtubule, even in a cell type (the developing spermatid) that simultaneously contains such functionally distinct structures as the manchette and the flagellum. On the other hand, the patterns of expression of the various tubulin isotypes show marked and distinctive differences in different cell types and, in at least one case, evidence is presented for regulation at the translational or posttranslational level. The significance of these observations is discussed in terms of the existence of the mammalian alpha- and beta-tubulin multigene families
— id: 11077, year: 1988, vol: 106, page: 2023, stat: Journal Article,

Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein
Lewis SA; Wang DH; Cowan NJ
1988 Nov 11;242(4880):936-939, Science
The microtubule-associated protein MAP2 is a prominent large-sized component of purified brain microtubules that, like the 36- to 38-kilodalton tau proteins, bears antigenic determinants found in association with the neurofibrillary tangles of Alzheimer's disease. The complete sequence of mouse brain MAP2 was determined from a series of overlapping cloned complementary DNAs. The sequence of the carboxyl-terminal 185 amino acids is very similar (67 percent) to a corresponding region of tau protein, and includes a series of three imperfect repeats, each 18 amino acids long and separated by 13 or 14 amino acids. A subcloned fragment spanning the first two of the 18-amino acid repeats was expressed as a polypeptide by translation in vitro. This polypeptide copurified with microtubules through two successive cycles of polymerization and depolymerization, whereas a control polypeptide derived from the amino-terminal region of MAP2 completely failed to copurify. These data imply that the carboxyl-terminal domain containing the 18-amino acid repeats constitutes the microtubule binding site in MAP2. The occurrence of these repeats in tau protein suggests that these may be a general feature of microtubule binding proteins
— id: 10886, year: 1988, vol: 242, page: 936, stat: Journal Article,

Complete sequence of a cDNA encoding mouse MAP2
Wang D; Lewis SA; Cowan NJ
1988 Dec 9;16(23):11369-11370, Nucleic acids research
— id: 10862, year: 1988, vol: 16, page: 11369, stat: Journal Article,

FUNCTION AND REGULATION OF EXPRESSION OF THE GENES ENCODING NEUROFILAMENT PROTEIN AND THE GLIAL FIBRILLARY ACIDIC PROTEIN
Cowan, NJ; Levy, E; Sarkan, S; Gordon, J; Lewis, S
1987 May 1;46(6):2145-2145, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 31361, year: 1987, vol: 46, page: 2145, stat: Journal Article,

Neurofilament gene expression: a major determinant of axonal caliber
Hoffman PN; Cleveland DW; Griffin JW; Landes PW; Cowan NJ; Price DL
1987 May;84(10):3472-3476, Proceedings of the National Academy of Sciences of the United States of America
Within the wide spectrum of axonal diameters occurring in mammalian nerve fibers, each class of neurons has a relatively restricted range of axonal calibers. The control of caliber has functional significance because diameter is the principal determinant of conduction velocity in myelinated nerve fibers. Previous observations support the hypothesis that neurofilaments (NF) are major intrinsic determinants of axonal caliber in large myelinated nerve fibers. Following interruption of axons (axotomy) by crushing or cutting a peripheral nerve, caliber is reduced in the proximal axonal stumps, which extend from the cell bodies to the site of axotomy. (The distal axonal stumps, which are disconnected from the cell bodies, degenerate and are replaced by the outgrowth of regenerating axonal sprouts arising from the proximal stump). This reduction in axonal caliber in the proximal stumps is associated with a selective diminution in the amount of NF protein undergoing slow axonal transport in these axons, with a decrease in axonal NF content, and with reduced conduction velocity. The present report demonstrates that changes in axonal caliber after axotomy correlate with a selective alteration in NF gene expression. Hybridization with specific cDNAs was used to measure levels of mRNA encoding the 68-kDa neurofilament protein (NF68), beta-tubulin, and actin in lumbar sensory neurons of rat at various times after crushing the sciatic nerve. Between 4 and 42 days after axotomy by nerve crush, the levels of NF68 mRNA were reduced 2- to 3-fold. At the same times, the levels of tubulin and actin mRNAs were increased several-fold. These findings support the hypothesis that the expression of a single set of neuron-specific genes (encoding NF) directly determines axonal caliber, a feature of neuronal morphology with important consequences for physiology and behavior
— id: 17143, year: 1987, vol: 84, page: 3472, stat: Journal Article,

Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein
Levy E; Liem RK; D'Eustachio P; Cowan NJ
1987 Jul 1;166(1):71-77, European journal of biochemistry
We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked
— id: 17141, year: 1987, vol: 166, page: 71, stat: Journal Article,

Free intermingling of mammalian beta-tubulin isotypes among functionally distinct microtubules
Lewis SA; Gu W; Cowan NJ
1987 May 22;49(4):539-548, Cell
Mammalian cells express a spectrum of tubulin isotypes whose relationship to the diversity of microtubule function is unknown. To examine whether different isotypes are segregated into functionally distinct microtubules, we generated immune sera capable of discriminating among the various naturally occurring beta-tubulin isotypes. Cloned fusion proteins encoding each isotype were used first to tolerogenize animals against shared epitopes, and then as immunogens to elicit a specific response. In experiments using these sera, we show that there is neither complete nor partial segregation of beta-tubulin isotypes: both interphase cytoskeletal and mitotic spindle microtubules are mixed copolymers of all expressed beta-tubulin isotypes. Indeed, a highly divergent isotype normally expressed only in certain hematopoietic cells is also indiscriminately assembled into all microtubules both in their normal context and when transfected into HeLa cells
— id: 17142, year: 1987, vol: 49, page: 539, stat: Journal Article,

Anomalous placement of introns in a member of the intermediate filament multigene family: an evolutionary conundrum
Lewis SA; Cowan NJ
1986 May;6(5):1529-1534, Molecular & cellular biology
The origin of introns and their role (if any) in gene expression, in the evolution of the genome, and in the generation of new expressed sequences are issues that are understood poorly, if at all. Multigene families provide a favorable opportunity for examining the evolutionary history of introns because it is possible to identify changes in intron placement and content since the divergence of family members from a common ancestral sequence. Here we report the complete sequence of the gene encoding the 68-kilodalton (kDa) neurofilament protein; the gene is a member of the intermediate filament multigene family that diverged over 600 million years ago. Five other members of this family (desmin, vimentin, glial fibrillary acidic protein, and type I and type II keratins) are encoded by genes with six or more introns at homologous positions. To our surprise, the number and placement of introns in the 68-kDa neurofilament protein gene were completely anomalous, with only three introns, none of which corresponded in position to introns in any characterized intermediate filament gene. This finding was all the more unexpected because comparative amino acid sequence data suggest a closer relationship of the 68-kDa neurofilament protein to desmin, vimentin, and glial fibrillary acidic protein than between any of these three proteins and the keratins. It appears likely that an mRNA-mediated transposition event was involved in the evolution of the 68-kDa neurofilament protein gene and that subsequent events led to the acquisition of at least two of the three introns present in the contemporary sequence
— id: 17148, year: 1986, vol: 6, page: 1529, stat: Journal Article,

Tubulin pseudogenes as markers for hominoid divergence
Lewis SA; Cowan NJ
1986 Feb 20;187(4):623-626, Journal of molecular biology
Processed pseudogenes arise via unimolecular events that result in the integration of nonfunctional (and therefore non-selected) regions of DNA into the germ line. The sequence of such pseudogenes can be used as a novel form of evolutionary clock: the older a particular pseudogene, the more mutations it has acquired relative to the selectively constrained functional gene from which it was originally derived. We have used specific beta-tubulin gene probes to assay for the presence of fully sequenced processed pseudogenes in genomic DNA from various hominoid species. The data suggest that orangutan is more closely related to human, chimpanzee and gorilla than is generally believed
— id: 17149, year: 1986, vol: 187, page: 623, stat: Journal Article,

A cloned cDNA encoding MAP1 detects a single copy gene in mouse and a brain-abundant RNA whose level decreases during development
Lewis SA; Sherline P; Cowan NJ
1986 Jun;102(6):2106-2114, Journal of cell biology
Screening of a bacteriophage lambda gt11 cDNA expression library with a polyclonal anti-microtubule associated protein (MAP) antiserum resulted in the isolation of two non-cross-hybridizing sets of cDNA clones. One set was shown to encode MAP2 (Lewis, S. A., A. Villasante, P. Sherline, and N. J. Cowan, 1986, J. Cell Biol., 102:2098-2105). To determine the specificity of the second set, three non-overlapping fragments cloned from the same mRNA molecule via a series of 'walking' experiments were separately subcloned into inducible plasmid expression vectors in the appropriate orientation and reading frame. Upon induction and analysis by immunoblotting, two of the fusion proteins synthesized were shown to be immunoreactive with an anti-MAP1-specific antibody, but not with an anti-MAP2-specific antibody. Since these MAP1-specific epitopes are encoded in non-overlapping cDNAs cloned from a single contiguous mRNA, these clones cannot encode polypeptides that contain adventitiously cross-reactive epitopes. Furthermore, these cDNA clones detected an abundant mRNA species of greater than 10 kb in mouse brain, consistent with the coding requirement of a 350,000-D polypeptide and the known abundance of MAP1 in that tissue. The MAP1-specific cDNA probes were used in blot transfer experiments with RNA prepared from brain, liver, kidney, stomach, spleen, and thymus. While detectable quantities of MAP1-specific mRNA were observed in these tissues, the level of MAP1 expression was approximately 500-fold lower than in brain. The levels of both MAP1-specific and MAP2-specific mRNAs decline in the postnatal developing brain; the level of MAP1-specific mRNA also increases slightly in rat PC12 cells upon exposure to nerve growth factor. These surprising results contrast sharply with reported dramatic developmental increases in the amount of MAP1 in brain and in nerve growth factor-induced PC12 cells. The cDNA clones encoding MAP1 detect a single copy sequence in mouse DNA, even under conditions of low stringency that would allow the detection of related but mismatched sequences. The cDNAs cross-hybridize with genomic sequences in rat, human, and chicken DNA, but not with DNA from frog, Drosophila, or sea urchin. These data are discussed in terms of the evolution and possible biological role of MAP1
— id: 17146, year: 1986, vol: 102, page: 2106, stat: Journal Article,

Brain-specific expression of MAP2 detected using a cloned cDNA probe
Lewis SA; Villasante A; Sherline P; Cowan NJ
1986 Jun;102(6):2098-2105, Journal of cell biology
We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA 'walking' experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2
— id: 17147, year: 1986, vol: 102, page: 2098, stat: Journal Article,

Six mouse alpha-tubulin mRNAs encode five distinct isotypes: testis-specific expression of two sister genes
Villasante A; Wang D; Dobner P; Dolph P; Lewis SA; Cowan NJ
1986 Jul;6(7):2409-2419, Molecular & cellular biology
Five mouse alpha-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the alpha-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (M alpha 3 and M alpha 7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of alpha-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that the duplication event leading to a pair of genes encoding a testis-specific alpha-tubulin isotype predated the mammalian radiation, and both members of the duplicated sequence have been maintained since species divergence. A second alpha-tubulin gene, M alpha 6, is expressed ubiquitously at a low level, whereas a third gene, M alpha 4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct alpha-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various alpha-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules
— id: 17145, year: 1986, vol: 6, page: 2409, stat: Journal Article,

The mammalian beta-tubulin repertoire: hematopoietic expression of a novel, heterologous beta-tubulin isotype
Wang D; Villasante A; Lewis SA; Cowan NJ
1986 Nov;103(5):1903-1910, Journal of cell biology
We describe the structure of a novel and unusually heterologous beta-tubulin isotype (M beta 1) isolated from a mouse bone marrow cDNA library, and a second isotype (M beta 3) isolated from a mouse testis cDNA library. Comparison of M beta 1 and M beta 3 with the completed (M beta 4, M beta 5) or extended (M beta 2) sequence of three previously described beta-tubulin isotypes shows that each includes a distinctive carboxy-terminal region, in addition to multiple amino acid substitutions throughout the polypeptide chain. In every case where a mammalian interspecies comparison can be made, both the carboxy-terminal and internal amino acid substitutions that distinguish one isotype from another are absolutely conserved. We conclude that these characteristic differences are important in determining functional distinctions between different kinds of microtubule. The amino acid homologies between M beta 2, M beta 3, M beta 4, and M beta 5 are in the range of 95-97%; however the homology between M beta 1 and all the other isotypes is very much less (78%). The dramatic divergence in M beta 1 is due to multiple changes that occur throughout the polypeptide chain. The overall level of expression of M beta 1 is low, and is restricted to those tissues (bone marrow, spleen, developing liver and lung) that are active in hematopoiesis in the mouse. We predict that the M beta 1 isotype is functionally specialized for assembly into the mammalian marginal band
— id: 17144, year: 1986, vol: 103, page: 1903, stat: Journal Article,

Structure of the mouse glial fibrillary acidic protein gene: implications for the evolution of the intermediate filament multigene family
Balcarek JM; Cowan NJ
1985 Aug 12;13(15):5527-5543, Nucleic acids research
We report the complete sequence of the gene encoding mouse glial fibrillary acidic protein (GFAP), the intermediate filament (IF) protein specific to astrocytes. The 9.8 kb gene includes nine exons separated by introns ranging in size from 0.2 to 2.5 kb. A comparison of the organization of the GFAP gene with that of genes encoding other IF proteins reveals that the structure of IF genes is highly conserved in spite of considerable divergence at the amino acid level. Thus, most of the evolutionary events leading to the placement of introns in IF genes must have occurred prior to the duplication and subsequent divergence of IF genes from a presumptive common ancestral sequence. The conserved gene organization is unrelated to structural features of IF proteins. A curious feature of the GFAP gene is the large number of repeated sequences found in the introns. Six tracts of reiterated di- or trinucleotides are present, plus tandem repeats of two different novel sequences. One repeat is unique to the GFAP gene; the other occurs elsewhere in the mouse genome, although at relatively low frequency
— id: 17152, year: 1985, vol: 13, page: 5527, stat: Journal Article,

Structural implications of a cDNA clone encoding mouse glial fibrillary acidic protein
Cowan NJ; Lewis SA; Balcarek JM; Krek V; Shelanski M
1985 ;455(1):575-582, Annals of the New York Academy of Sciences
— id: 17156, year: 1985, vol: 455, page: 575, stat: Journal Article,

THE PHONOLOGICAL AND METAPHONOLOGICAL REPRESENTATION OF SPEECH - EVIDENCE FROM FLUENT BACKWARD TALKERS
COWAN, N; BRAINE, MDS; LEAVITT, LA
1985 JAN 20 ;24(6):679-698, Journal of memory & language
— id: 98566, year: 1985, vol: 24, page: 679, stat: Journal Article,

Structural features and restricted expression of a human alpha-tubulin gene
Hall JL; Cowan NJ
1985 Jan 11;13(1):207-223, Nucleic acids research
The nucleotide sequence of a human alpha-tubulin gene (b alpha 1) is described. This gene is extensively homologous to a rat alpha-tubulin gene in its coding regions, 3'-untranslated region and, indeed, in segments of its largest intron. However, with the exception of three short conserved blocks of homology, the 5' flanking regions of the rat and human genes are unrelated. Hence, these genes each encoding an identical protein are transcribed under the influence of divergent promoters. Blot analyses using RNA from a variety of transformed cells derived from different tissues indicate that expression of the human alpha-tubulin gene is restricted to cells of neurological origin. Among neurological cell types b alpha 1 expression is further restricted to adherent cells that are morphologically differentiated. The data presented suggest that the b alpha 1 gene encodes a prominent neuronal and glial alpha-tubulin and that b alpha 1 expression is a function of the differentiated state of these cells
— id: 17155, year: 1985, vol: 13, page: 207, stat: Journal Article,

Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe
Lewis SA; Cowan NJ
1985 Mar;100(3):843-850, Journal of cell biology
A 1.2-kilobase (kb) cDNA clone (NF68) encoding the mouse 68,000-mol-wt neurofilament protein is described. The clone was isolated from a mouse brain cDNA library by low-stringency cross-hybridization with a cDNA probe encoding mouse glial fibrillary acidic protein (Lewis et al., 1984, Proc. Natl. Acad. Sci. USA., 81:2743-2746). The identity of NF68 was established by hybrid selection using mouse brain polyA+ mRNA, and cell-free translation of the selected mRNA species. The cell-free translation product co-migrated with authentic 68,000-mol-wt neurofilament protein on an SDS/polyacrylamide gel, and was immunoprecipitable with a monospecific rabbit anti-bovine neurofilament antiserum. In addition, DNA sequence analysis of NF68 showed 90% homology at the amino acid level compared with the sequence of the porcine 68,000-mol-wt neurofilament protein. At high stringency, NF68 detects a single genomic sequence encoding the mouse 68,000-mol-wt neurofilament protein. Two mRNA species of 2.5 kb and 4.0 kb are transcribed from the single gene in mouse brain. The level of expression of these mRNAs remains almost constant in postnatal mouse brains of all ages and, indeed, in the adult. At reduced stringency, NF68 detects a number of mRNAs that are expressed in mouse brain, one of which encodes the 150,000-mol-wt neurofilament protein. The NF68 probe cross-hybridizes at high stringency with genomic sequences in species as diverse as human, chicken, and (weakly) frog, but not with DNA from Drosophila or sea urchin
— id: 17154, year: 1985, vol: 100, page: 843, stat: Journal Article,

Temporal expression of mouse glial fibrillary acidic protein mRNA studied by a rapid in situ hybridization procedure
Lewis SA; Cowan NJ
1985 Sep;45(3):913-919, Journal of neurochemistry
A rapid and sensitive in situ hybridization technique is described for the detection of mRNA sequences in 6-8-micron cryostat sections. The method incorporates the use of alpha-thio-35S-labelled nucleoside triphosphates for the generation of high-specific-activity DNA probes and a high-stringency washing procedure that virtually eliminates background without unduly compromising histological integrity. Whereas signal resolution is less than that observed using 3H probes, 35S-labelled probes are well-suited for experiments where resolution at the cellular level is required. The method has been applied to a study of the developmental regulation of glial fibrillary acidic protein (GFAP) mRNA expression in developing mouse brain. GFAP-specific sequences are first detectable after the second postnatal day, and thereafter rise to a level that is maintained throughout development and into adulthood. The distribution of GFAP-encoding sequences broadly reflects the known distribution of astrocytes, but the levels of mRNA within these cells vary by a surprisingly large amount depending on their location. For example, in adult animals, the astrocytes of the glial limitans contain an abundance of GFAP-specific mRNA that is higher than corresponding levels in astrocytes in the cerebellar white matter, whereas these cells in turn contain considerably more GFAP-specific mRNA than astrocytes in the gray matter of the cerebrum. Unexpectedly, parallel RNA blot transfer experiments show the existence of some GFAP-encoding mRNA size heterogeneity that is restricted to the first postnatal week
— id: 17151, year: 1985, vol: 45, page: 913, stat: Journal Article,

Three expressed sequences within the human beta-tubulin multigene family each define a distinct isotype
Lewis SA; Gilmartin ME; Hall JL; Cowan NJ
1985 Mar 5;182(1):11-20, Journal of molecular biology
This paper describes the isolation and complete sequence of a novel expressed human beta-tubulin gene (beta 2). The sequence is compared with that of two other expressed human beta-tubulin genes (M40 and 5 beta). All are encoded by four exons. Though the boundaries of each exon are absolutely conserved among the three genes, the intervening sequences differ considerably in size and sequence content. Two of the genes (M40 and 5 beta) contain one (M40) or ten (5 beta) members of the middle repetitive Alu family sequences within one of their intervening sequences. Comparison of the amino acid sequences encoded by each gene reveals a high level of homology overall, though there is significant divergence between the carboxy termini of two of the genes. The pattern of expression of each beta-tubulin gene has been studied in several different human cell lines using unique non-crosshybridizing probes derived from the 3' untranslated regions. Two of the genes, M40 and beta 2, are expressed at varying levels in all of the cell lines examined, though the level of expression of one of these genes parallels the other in most cases. The third gene, 5 beta, is detectably expressed only in cells of neural origin. Thus, distinct human beta-tubulin isotypes are encoded by genes whose exon size and number has been conserved evolutionarily, but whose pattern of expression may be regulated either co-ordinately or uniquely. Of the approximately 15 sequences contained in the human beta-tubulin multigene family, nine have now been sequenced fully. The overall composition of the multigene family and the evolutionary relationships among its various members are discussed
— id: 17153, year: 1985, vol: 182, page: 11, stat: Journal Article,

Five mouse tubulin isotypes and their regulated expression during development
Lewis SA; Lee MG; Cowan NJ
1985 Sep;101(3):852-861, Journal of cell biology
We describe five mouse tubulin cloned cDNAs, two (M alpha 1 and M alpha 2) that encode alpha-tubulin and three (M beta 2, M beta 4, and M beta 5) that encode beta-tubulin. The sequence of these clones reveals that each represents a distinct gene product. Within the sequence common to the two alpha-tubulin cDNAs, the encoded amino acids are identical, though the 3' noncoding regions are wholly dissimilar. In contrast, the three beta-tubulin cDNAs show considerable carboxy-terminal heterogeneity. Two of the beta-tubulin isotypes defined by the cloned sequences are absolutely conserved between mouse and human, and all three beta-tubulin isotypes are conserved between mouse and rat. This result implies the existence of selective constraints that have maintained sequence identity after species divergence. This conclusion is reinforced by the near identity between a third mouse beta-tubulin isotype and a chicken beta-tubulin isotype. The significance of the interspecies conservation of tubulin isotypes is discussed in relationship to microtubule function. We have used non-cross-hybridizing 3' noncoding region probes from the five cloned mouse tubulin cDNAs to study the developmental expression of each isotype in various mouse tissues. M alpha 1 and M beta 2 are expressed in an approximately coordinate fashion, and their transcripts are most abundant in brain and lung. M alpha 2 and M beta 5 are ubiquitously expressed and to a similar extent in each tissue, with the greatest abundance in spleen, thymus, and immature brain. In contrast, M beta 4 is expressed exclusively in brain. Whereas the expression of the latter isotype increases dramatically during postnatal development, transcripts from all four other tubulin genes decline from maximum levels at or before birth. Tissue-specific development changes in the abundance of tubulin isotype-specific mRNAs are discussed in relationship to organogenesis in the mouse
— id: 17150, year: 1985, vol: 101, page: 852, stat: Journal Article,

Tubulin genes and the diversity of microtubule function
Cowan NJ
1984 ;1(9):36-60, Oxford surveys on eukaryotic genes
— id: 17159, year: 1984, vol: 1, page: 36, stat: Journal Article,

SALIENT CHILDHOOD MEMORIES
COWAN, N; DAVIDSON, G
1984 JAN 20 ;145(1):101-107, Journal of genetic psychology
— id: 98589, year: 1984, vol: 145, page: 101, stat: Journal Article,

Sequence of an expressed human beta-tubulin gene containing ten Alu family members
Lee MG; Loomis C; Cowan NJ
1984 Jul 25;12(14):5823-5836, Nucleic acids research
The complete sequence of a functionally expressed human beta-tubulin gene (5 beta) is presented. The amino acid sequence encoded by this gene constitutes a distinct isotype, differing from a previously described human beta-tubulin sequence at 21 positions throughout the polypeptide chain. The beta-tubulin coding sequence in 5 beta is interrupted by three intervening sequences of 1014, 117 and 4826 nucleotides. The largest of these contains ten members of the Alu family of middle repetitive sequences. Together, these regions account for sixty percent of this intervening sequence. Two of the Alu elements are juxtaposed head to tail, and share the same flanking direct repeat. The ten Alu sequences are substantially divergent, both from each other and from an Alu consensus sequence, and several contain deletions of up to half the entire sequence
— id: 17157, year: 1984, vol: 12, page: 5823, stat: Journal Article,

Sequence of a cDNA clone encoding mouse glial fibrillary acidic protein: structural conservation of intermediate filaments
Lewis SA; Balcarek JM; Krek V; Shelanski M; Cowan NJ
1984 May;81(9):2743-2746, Proceedings of the National Academy of Sciences of the United States of America
A clone encoding mouse glial fibrillary acidic protein (GFAP) was isolated from a cDNA library constructed so as to express the cloned sequences. The library was screened using a GFAP-specific polyclonal antiserum; a single bacterial colony expressing GFAP was identified. The complete sequence of the cDNA insert in this clone is presented, encompassing 2.5 kilobases and specifying greater than 97% of the GFAP amino acid sequence. The clone includes a long (1.4-kilobase) 3' untranslated region. Within the coding region, the data show extensive homology with other intermediate filament proteins, particularly in those regions predicted to be alpha-helical. RNA blot transfer experiments using the cloned GFAP cDNA probe revealed a single GFAP mRNA species of 2.7 kilobases in mouse brain. Southern blot analysis indicates the existence of at most two genes encoding GFAP in the mouse genome. The mouse GFAP probe cross-hybridizes weakly at high stringency with genomic DNA from diverse eukaryotic species
— id: 17158, year: 1984, vol: 81, page: 2743, stat: Journal Article,

Expression of human alpha-tubulin genes: interspecies conservation of 3' untranslated regions
Cowan NJ; Dobner PR; Fuchs EV; Cleveland DW
1983 Oct;3(10):1738-1745, Molecular & cellular biology
To examine the sequence complexity and differential expression of human alpha-tubulin genes, we constructed cDNA libraries from two unrelated tissue types (epidermis and fetal brain). The complete sequence of a positively hybridizing alpha-tubulin clone from each library is described. Each is shown to represent an abundantly expressed gene from fetal brain and keratinocytes, respectively. Although the coding regions are extensively homologous (97%), the 3' untranslated regions are totally dissimilar. This property has been used to dissect the human alpha-tubulin multigene family into members bearing sequence relatedness in this region. Surprisingly, each of these noncoding regions shares very high (65 to 80%) interspecies homology with the 3' untranslated region of one of the two rat alpha-tubulin genes of known sequence. These unexpected homologies imply the existence of selective pressure on the 3' untranslated regions of some cytoskeletal genes which maintains sequence fidelity during the course of evolution, perhaps as a consequence of an as yet unidentified functional requirement
— id: 17160, year: 1983, vol: 3, page: 1738, stat: Journal Article,

Tubulin isotypes and the multigene tubulin families
Cowan NJ; Dudley L
1983 ;85(5):147-173, International review of cytology
— id: 17163, year: 1983, vol: 85, page: 147, stat: Journal Article,

Identification of two human beta-tubulin isotypes
Hall JL; Dudley L; Dobner PR; Lewis SA; Cowan NJ
1983 May;3(5):854-862, Molecular & cellular biology
The sequence of a human beta-tubulin cDNA clone (D beta-1) is described; our data revealed 95.6% homology compared with the sequence of a human beta-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken beta-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human beta-tubulin sequence (5 beta) possessed a very high degree of homology with chicken and pig beta-tubulins in this region. Thus, human cells appear to contain two distinct beta-tubulin isotypes. Both the intact beta-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 5 beta genomic sequence also detected a 2.6-kilobase beta-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human beta-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating beta-tubulin mRNAs
— id: 17162, year: 1983, vol: 3, page: 854, stat: Journal Article,

Evolutionary history of a multigene family: an expressed human beta-tubulin gene and three processed pseudogenes
Lee MG; Lewis SA; Wilde CD; Cowan NJ
1983 Jun;33(2):477-487, Cell
A 3' untranslated region subclone from a human beta-tubulin cDNA clone has been used to dissect the human beta-tubulin multigene family. Four different beta-tubulin sequences were obtained. One consists of an expressed gene that yields two mRNA species of 1.8 kb and 2.6 kb as a consequence of alternative polyadenylation sites. The three remaining beta-tubulin sequences are all intronless pseudogenes, each containing a 3' poly(A) tract downstream from the poly(A) signal, and each flanked by a different short direct repeat. Two of these sequences were derived by integration into the host germ line of cDNA copies of the 1.8 kb mRNA; the third was derived from the 2.6 kb mRNA. Comparison of the functional and nonfunctional sequences suggests that the integration events took place 4, 10, and 13 million years ago. We anticipate that, in multigene families where germ-line expression occurs, a significant portion of sequences will be accounted for by pseudogenes generated via an RNA intermediate
— id: 17161, year: 1983, vol: 33, page: 477, stat: Journal Article,

Structure of two human alpha-tubulin genes
Wilde CD; Chow LT; Wefald FC; Cowan NJ
1982 Jan;79(1):96-100, Proceedings of the National Academy of Sciences of the United States of America
The ability of a chicken alpha-tubulin cDNA probe to cross-hybridize with human DNA under stringent conditions has been exploited to screen two independently constructed human genomic libraries. Nine clones were isolated, accounting for 60% of the bands observed in a whole genomic Southern blot of human DNA. Two clones were selected for further analysis by restriction mapping, orientation experiments using 3'- or 5'-specific probes, and electron microscopy of heteroduplexes. One clone, 2 alpha, contains an alpha-tubulin-specific region of 5.0 kilobases that includes three intervening sequences. The second clone, 19 alpha, contains an alpha-tubulin-specific region of 5.4 kilobases and has somewhat diverged 5' and 3' ends. Clone 19 alpha has only two intervening sequences that correspond to the first two in clone 2 alpha. However, these intervening sequences differ in size between clones 2 alpha and 19 alpha and show no detectable sequence homology. The sum of the lengths of sequences in either clone that hybridize to the cDNA probe accounts for essentially the entire length of the cDNA molecule
— id: 17167, year: 1982, vol: 79, page: 96, stat: Journal Article,

Diverse mechanisms in the generation of human beta-tubulin pseudogenes
Wilde CD; Crowther CE; Cowan NJ
1982 Aug 6;217(4559):549-549, Science
The sequence of two human beta-tubulin pseudogenes is described. One contains an intervening sequence but lacks sequences encoding the 55 N-terminal amino acids of the polypeptide chain. A second has no introns but has a polyadenylate signal and an oligoadenylate tract at its 3' end, and it is flanked by a short direct repeat. These sequences have arisen by different mechanisms, including one that probably involves reverse transcription of a processed messenger RNA and reintegration of the complementary DNA copy into the genome
— id: 17164, year: 1982, vol: 217, page: 549, stat: Journal Article,

Isolation of a multigene family containing human alpha-tubulin sequences
Wilde CD; Crowther CE; Cowan NJ
1982 Mar 15;155(4):533-538, Journal of molecular biology
— id: 17166, year: 1982, vol: 155, page: 533, stat: Journal Article,

Evidence that a human beta-tubulin pseudogene is derived from its corresponding mRNA
Wilde CD; Crowther CE; Cripe TP; Gwo-Shu Lee M; Cowan NJ
1982 May 6;297(5861):83-84, Nature
— id: 17165, year: 1982, vol: 297, page: 83, stat: Journal Article,

Structural variation among human beta-tubulin genes
Cowan NJ; Wilde CD; Chow LT; Wefald FC
1981 Aug;78(8):4877-4881, Proceedings of the National Academy of Sciences of the United States of America
A chicken beta-tubulin cDNA probe has been used to screen two independently generated human genomic libraries. Of 13 EcoRI fragments detectable in a human genomic Southern blot experiment, 7 correspond in size to EcoRI fragments isolated from recombinant bacteriophage. The location of beta-tubulin-specific regions and the direction of transcription were determined within each cloned fragment. One clone (5 beta) contained a beta-tubulin-specific region of 6.8 kilobase pairs (kbp) that included three intervening sequences as well as a number of inverted repeat structures. The remaining clones contained beta-tubulin-specific sequences that were close to or, in two cases, substantially less than 1.9 kbp long. Because mature human beta-tubulin mRNA is approximately 1.9 kbp long, these short DNA regions cannot on their own encode a functional beta-tubulin mRNA. Analysis using 3'- and 5'-specific probes derived from the chicken cDNA clone showed the presence of both of these end regions within one truncated tubulin-like sequence. A second short tubulin-specific region failed to hybridize with a 3'-specific probe. These short sequences are therefore likely to be examples of pseudogenes that have arisen by loss of a portion of DNA essential to the production of functional human beta-tubulin mRNA
— id: 17168, year: 1981, vol: 78, page: 4877, stat: Journal Article,

Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes
Cleveland DW; Lopata MA; MacDonald RJ; Cowan NJ; Rutter WJ; Kirschner MW
1980 May;20(1):95-105, Cell
— id: 17169, year: 1980, vol: 20, page: 95, stat: Journal Article,

Isolation of separate mRNAs for alpha- and beta-tubulin and characterization of the corresponding in vitro translation products
Cleveland DW; Kirschner MW; Cowan NJ
1978 Nov;15(3):1021-1031, Cell
The messenger RNAs coding for alpha- and beta-tubulin have been isolated from embryonic chick brain. Although the mRNAs for the two tubulin subunits have been resolved on native gels, they are very similar in molecular weight (650,000 daltons) as judged by mobility on denaturing gels containing methy mercury. The mRNAs for beta- and gamma-actin have also been resolved on native gels, but migrate as an unresolved peak (molecular weight 6500,000-700,000 daltons) under denaturing conditions. Since the nonmuscle actins are substantially smaller proteins than alpha- and beta-tubulin, the large size of chick nonmuscle actin mRNAs suggests an unusually long untranslated region. Since tubulin and actin polypeptides are internal structural proteins, one would expect them to be synthesized only on free polysomes. Translation of mRNA derived directly from a purified membrane fraction or by puromycin release from that fraction, however, showed the synthesis of a small proportion of these proteins on polysomes that are membrane-associated. Peptide mapping has in all cases confirmed the identity of the products of cell-free synthesis with authentic alpha-tubulin, beta-tubulin and actin. Approximately 67% of the alpha- and 13% of the beta-tubulin chains produced by in vitro translation are competent for co-assembly into microtubules with added carrier microtubule protein
— id: 17170, year: 1978, vol: 15, page: 1021, stat: Journal Article,

Expression of antibody genes in tissue culture: structural mutants and hybrid cells
Milstein C; Adetugbo K; Cowan NJ; Kohler G; Secher DS
1978 May;15(48):321-330, National Cancer Institute monograph
Detailed information on the nature and frequency of somatic mutations has been derived from studies of the clonal diversification of the myeloma MOPC 21 in tissue culture. A screening procedure is described that permitted the isolation of four spontaneous mutations at the gamma1 structural gene locus. These originate from four mutation events. Two seem to be point mutations: a 'nonsense' and a 'mis-sense.' Of the other two, one is a frameshift leading to mistranslation and early termination, the other a large deletion due to perhaps an intrachromosomal translocation or a mitotic recombination. Fusion between myeloma-producing cells has shown that variable and constant region genes cannot be scrambled. Differentiation from stem to plasma cells seems to involve changes in the primary sequence of the DNA. Fusion between myeloma cells and spleen cells from immunized animals is a satisfactory method for the derivation of permanent tissue culture lines producing specific antibody. The hybrids express the myeloma as well as the specific antibody light and heavy chains. By subcloning and selection, one can derive lines that selectively lose individual chains. Lines that no longer express the myeloma components can thus be derived. The use of appropriate defective variants of the myeloma parental line is another way of avoiding the presence of the myeloma components
— id: 17171, year: 1978, vol: 15, page: 321, stat: Journal Article,

Somatic cell genetics of antibody-secreting cells: studies of clonal diversification and analysis by cell fusion
Milstein C; Adetugbo K; Cowan NJ; Kohler G; Secher DS; Wilde CD
1977 ;41 Pt 2(48):793-803, Cold Spring Harbor symposia on quantitative biology
— id: 17172, year: 1977, vol: 41 Pt 2, page: 793, stat: Journal Article,

Purification and sequence analysis of the mRNA coding for an immunoglobulin heavy chain
Cowan NJ; Secher DS; Milstein C
1976 Jan 15;61(2):353-368, European journal of biochemistry
A mutant cell line (IF2) derived from the mouse myeloma MOPC 21 has been used for the isolation and sequence analysis of H-chain mRNA. The IF2 cells synthesise an H-chain of reduced size in which the CH1 homology region is missing. Sizing of the IF2 H-chain mRNA and wild-type H-chain mRNA revealed that the deletion is expressed at the mRNA level. The mutant H-chain mRNA sedimented at 16-S, enabling effective resolution from 18-S ribosomal RNA. In experiments using IF2 cells labelled with [32P]phosphate, the 16-S mRNA was purified by oligo(T)-cellulose chromatography. Polyacrylamide gel analysis of the poly(A)-containing fraction showed the presence of a single radioactive band. Comparison of the mobility of this band relative to markers of known molecular weight revealed that the molecule contained about 1600 nucleotides. Digestion of the 32-P-labelled mRNA with T1 ribonuclease and two-dimensional fractionation of the resulting oligonucleotides yielded a 'finger-print' suitable for a preliminary sequence analysis. By using the established amino acid sequence of the IF2 H-chain and a knowledge of the genetic code, 14 oligonucleotides were assigned within the constant region and four within the variable region of the IF2 H-chain. This sequence data accounts for 19.5% of the coding region. Several other oligonucleotides, which could not be assigned within the coding region but which occurred in approximately molar yield, have also been partially characterised. These oligonucleotides are presumably derived from the untranslated regions of mRNA
— id: 17173, year: 1976, vol: 61, page: 353, stat: Journal Article,

Stability of cytoplasmic ribonucleic acid in a mouse myeloma: estimation of the half-life of the messenger RNA coding for an immunoglobulin light chain
Cowan NJ; Milstein C
1974 Feb 5;82(4):469-481, Journal of molecular biology
— id: 17175, year: 1974, vol: 82, page: 469, stat: Journal Article,

Intracellular immunoglobulin chain synthesis in non-secreting variants of a mouse myeloma: detection of inactive light-chain messenger RNA
Cowan NJ; Secher DS; Milstein C
1974 Dec 25;90(4):691-701, Journal of molecular biology
— id: 17174, year: 1974, vol: 90, page: 691, stat: Journal Article,

Purification and sequence of messenger RNA for immunoglobulin light chains
Brownlee GG; Cartwright EM; Cowan NJ; Jarvis JM; Milstein C
1973 Aug 22;244(138):236-240, Nature: new biology
— id: 17176, year: 1973, vol: 244, page: 236, stat: Journal Article,

The translation in vitro of mRNA for immunoglobulin heavy chains
Cowan NJ; Milstein C
1973 Jul 2;36(1):1-7, European journal of biochemistry
— id: 17177, year: 1973, vol: 36, page: 1, stat: Journal Article,

The secretion of a sialic acid-free immunoglobulin
Cowan NJ; Secher DS; Cotton RG; Milstein C
1973 Mar 15;30(3):343-346, FEBS letters
— id: 17178, year: 1973, vol: 30, page: 343, stat: Journal Article,

Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells
Cowan NJ; Milstein C
1972 Jun;128(2):445-454, Biochemical journal
— id: 17179, year: 1972, vol: 128, page: 445, stat: Journal Article,

The sequence of addition of terminal sugars to an immunoglobulin A myeloma protein
Cowan NJ; Robinson GB
1972 Feb;126(3):751-754, Biochemical journal
— id: 17180, year: 1972, vol: 126, page: 751, stat: Journal Article,

The subcellular acquisition of carbohydrates by glycoproteins
Cowan NJ; Robinson GB
1969 Dec;115(5):57P-57P, Biochemical journal
— id: 17181, year: 1969, vol: 115, page: 57P, stat: Journal Article,