The cloning of zebrin II reveals its identity with aldolase C

Development ◽  
1994 ◽  
Vol 120 (8) ◽  
pp. 2081-2090 ◽  
Author(s):  
A.H. Ahn ◽  
S. Dziennis ◽  
R. Hawkes ◽  
K. Herrup
Keyword(s):  
Purkinje Cells ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Aldolase C ◽  
Zebrin Ii ◽  
Antibody Staining ◽  
Afferent Projections ◽  
Biochemical Level ◽  
Cerebellar Purkinje Cells

The sagittal organization of the mammalian cerebellum can be observed at the anatomical, physiological and biochemical level. Previous screening of monoclonal antibodies produced in our laboratory has identified two intracellular antigens, zebrin I and II, that occur exclusively in adult cerebellar Purkinje cells. As their name suggests, the zebrin antibody staining of the Purkinje cell population is not uniform. Rather, zebrin-positive Purkinje cells are organized in stripes or bands that run from anterior to posterior across most of the cerebellum; interposed between the zebrin-positive cells are bands of Purkinje cells that are zebrin-negative. Comparison of the position of the antigenic bands with the anatomy of afferent projections suggests that the bands are congruent with the basic developmental and functional ‘compartments’ of the cerebellum. We report the isolation of cDNA clones of the 36 × 10(3) M(r) antigen, zebrin II, by screening of a mouse cerebellum cDNA expression library. Sequence analysis reveals a 98% identity between our clone and the glycolytic isozyme, aldolase C. In order to more rigorously demonstrate the identity of the two proteins, we stained adult cerebellum with an independent monoclonal antibody raised against aldolase C. Anti-aldolase staining occurs in a previously unreported pattern of sagittal bands of Purkinje cells; the pattern is identical to that revealed by the zebrin II monoclonal. Further, in situ hybridization of antisense aldolase C riboprobe shows that the accumulation of zebrin II/aldolase C mRNA corresponds to the pattern of the zebrin antigen in Purkinje cells. Zebrin II/aldolase C gene expression is thus regulated at the level of transcription (or mRNA stability). In light of previous work that has demonstrated the cell-autonomous and developmentally regimented expression of zebrin II, further studies of the regulation of this gene may lead to insights about the determination of cerebellar compartmentation.

Zebrin Expression in the Cerebellum of Two Crocodilian Species

2020 ◽  
Vol 95 (1) ◽  
pp. 45-55
Author(s):  
Cristián  Gutiérrez-Ibáñez  ◽  
Max R. Dannish ◽  
Tobias Kohl ◽  
Lutz  Kettler ◽  
Catherine E. Carr ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Purkinje Cells ◽  
Glycolytic Enzyme ◽  
Alligator Mississippiensis ◽  
Aldolase C ◽  
Zebrin Ii ◽  
Close Phylogenetic Relationship ◽  
Afferent Inputs ◽  
Sagittal Zones ◽  
Cerebellar Purkinje Cells

While in birds and mammals the cerebellum is a highly convoluted structure that consists of numerous transverse lobules, in most amphibians and reptiles it consists of only a single unfolded sheet. Orthogonal to the lobules, the cerebellum is comprised of sagittal zones that are revealed in the pattern of afferent inputs, the projection patterns of Purkinje cells, and Purkinje cell response properties, among other features. The expression of several molecular markers, such as aldolase C, is also parasagittally organized. Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme expressed in the cerebellar Purkinje cells of the vertebrate cerebellum. In birds, mammals, and some lizards (Ctenophoresspp.), ZII is expressed in a heterogenous fashion of alternating sagittal bands of high (ZII+) and low (ZII–) expression Purkinje cells. In contrast, turtles and snakes express ZII homogenously (ZII+) in their cerebella, but the pattern in crocodilians is unknown. Here, we examined the expression of ZII in two crocodilian species (Crocodylus niloticus and Alligator mississippiensis) to help determine the evolutionary origin of striped ZII expression in vertebrates. We expected crocodilians to express ZII in a striped (ZII+/ZII–) manner because of their close phylogenetic relationship to birds and their larger and more folded cerebellum compared to that of snakes and turtles. Contrary to our prediction, all Purkinje cells in the crocodilian cerebellum had a generally homogenous expression of ZII (ZII+) rather than clear ZII+/– stripes. Our results suggest that either ZII stripes were lost in three groups (snakes, turtles, and crocodilians) or ZII stripes evolved independently three times (lizards, birds, and mammals).

Zebrin II Is Expressed in Sagittal Stripes in the Cerebellum of Dragon Lizards (Ctenophorus sp.)

2016 ◽  
Vol 88 (3-4) ◽  
pp. 177-186 ◽  
Author(s):  
Douglas R. Wylie ◽  
Daniel Hoops ◽  
Joel W. Aspden ◽  
Andrew N. Iwaniuk
Keyword(s):  
Purkinje Cells ◽  
Posterior Part ◽  
Glycolytic Enzyme ◽  
Ancestral Condition ◽  
Aldolase C ◽  
Evolutionary Origins ◽  
Zebrin Ii ◽  
Heterogeneous Expression ◽  
Cerebellar Purkinje Cells

Aldolase C, also known as zebrin II (ZII), is a glycolytic enzyme that is expressed in cerebellar Purkinje cells of the vertebrate cerebellum. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). In contrast, in snakes and turtles, ZII is not expressed heterogeneously; rather all Purkinje cells are ZII+. Here, we examined the expression of ZII in the cerebellum of lizards to elucidate the evolutionary origins of ZII stripes in Sauropsida. We focused on the central netted dragon (Ctenophorus nuchalis) but also examined cerebellar ZII expression in 5 other dragon species (Ctenophorus spp.). In contrast to what has been observed in snakes and turtles, we found that in these lizards, ZII is heterogeneously expressed. In the posterior part of the cerebellum, on each side of the midline, there were 3 sagittal stripes consisting of Purkinje cells with high ZII expression (ZII+) alternating with 2 sagittal stripes with weaker ZII expression (ZIIw). More anteriorly, most of the Purkinje cells were ZII+, except laterally, where the Purkinje cells did not express ZII (ZII-). Finally, all Purkinje cells in the auricle (flocculus) were ZII-. Overall, the parasagittal heterogeneous expression of ZII in the cerebellum of lizards is similar to that in mammals and birds, and contrasts with the homogenous ZII+ expression seen in snakes and turtles. We suggest that a sagittal heterogeneous expression of ZII represents the ancestral condition in stem reptiles which was lost in snakes and turtles.

scholarly journals A novel 205-kilodalton testis-specific serine/threonine protein kinase associated with microtubules of the spermatid manchette.

1993 ◽  
Vol 13 (12) ◽  
pp. 7625-7635 ◽  
Author(s):  
P D Walden ◽  
N J Cowan
Keyword(s):  
Protein Complex ◽  
Catalytic Domain ◽  
Signal Transduction Pathway ◽  
Kinase Domain ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Serine Threonine Kinase ◽  
Expression Clone

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.

Role of Amplified Genes in the Production of Autoantibodies

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2158-2166 ◽  
Author(s):  
Nicole Brass ◽  
Alexander Rácz ◽  
Christine Bauer ◽  
Dirk Heckel ◽  
Gerhard Sybrecht ◽  
...  
Keyword(s):  
Gene Amplification ◽  
Squamous Cell ◽  
Lung Carcinoma ◽  
Chromosomal Abnormalities ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Squamous Cell Lung Carcinoma ◽  
Expression Library ◽  
Cell Lung Carcinoma

Abstract A variety of previously published studies have shown the presence of autoantibodies directed against oncogenic proteins in the sera of patients with tumors. Generally the underlying genetic aberration responsible for the induction of an immune response directed against an abnormal protein is unknown. In our studies we analyzed the role of gene amplification in the production of autoantibodies in squamous cell lung carcinoma. We screened a cDNA expression library with autologous patient serum and characterized the isolated cDNA clones encoding tumor expressed antigens termed LCEA (lung carcinoma expressed antigens). As determined by sequence analysis, the 35 identified cDNA clones represent 19 different genes of both known and unknown function. The spectrum of different clones were mapped by polymerase chain reaction (PCR) and fluorescence in-situ hybridization, showing that a majority are located on chromosome 3, which is frequently affected by chromosomal abnormalities in lung cancer. Gene amplification of 14 genes was analyzed by comparative PCR. Nine genes (65% of all analyzed genes) were found to be amplified; furthermore, most of them are also overrepresented in the pool of cDNA clones, suggesting an overexpression in the corresponding tumor. These results strongly suggest that gene amplification is one possible mechanism for the expression of immunoreactive antigens in squamous cell lung carcinoma.

scholarly journals Structural features of the low-molecular-mass human salivary mucin

1992 ◽  
Vol 287 (2) ◽  
pp. 639-643 ◽  
Author(s):  
M S Reddy ◽  
L A Bobek ◽  
G G Haraszthy ◽  
A R Biesbrock ◽  
M J Levine
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Structural Features ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mrna Species ◽  
Salivary Mucin

The low-molecular-mass human salivary mucin has at least two isoforms, MG2a and MG2b, that differ primarily in their sialic acid and fucose content. In this study, we characterize further these isoforms, particularly their peptide moieties. Trypsin digests of MG2a and MG2b yielded high- and low-molecular-mass glycopeptides following gel filtration on Sephacryl S-300. The larger glycopeptides from MG2a and MG2b had similar amino acid compositions and identical N-terminal sequences, suggesting common structural features between their peptides. An oligonucleotide probe generated from the amino acid sequence of the smaller glycopeptide from MG2a was employed in Northern-blot analysis. This probe specifically hybridized to two mRNA species from human submandibular and sublingual glands. A cDNA clone selected from a human submandibular gland cDNA expression library with antibody generated against deglycosylated MG2a also hybridized to these two mRNA species. In both cases, the larger mRNA was polydisperse, and the hybridization signal was more intense in the sublingual gland. In addition, the N-terminal amino acid sequence of the larger glycopeptide was found to be part of one of the selected MG2 cDNA clones.

scholarly journals NuMA: an unusually long coiled-coil related protein in the mammalian nucleus.

1992 ◽  
Vol 116 (6) ◽  
pp. 1303-1317 ◽  
Author(s):  
C H Yang ◽  
E J Lambie ◽  
M Snyder
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Nuclear Lamina ◽  
Early Prophase ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Mitotic Apparatus ◽  
Reading Frame ◽  
Nuclear Lamins

A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.

scholarly journals Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38)

1990 ◽  
Vol 270 (1) ◽  
pp. 97-102 ◽  
Author(s):  
J P Luzio ◽  
B Brake ◽  
G Banting ◽  
K E Howell ◽  
P Braghetta ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Integral Membrane Protein ◽  
Polyclonal Antiserum ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Expression Library ◽  
Trans Golgi Network ◽  
Sequencing And Expression ◽  
Novel Strategy ◽  
Golgi Network

Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network.

Zebrin II Expression in the Cerebellum of a Paleognathous Bird, the Chilean Tinamou (Nothoprocta perdicaria)

2015 ◽  
Vol 85 (2) ◽  
pp. 94-106 ◽  
Author(s):  
Jeremy R. Corfield ◽  
Jeffery Kolominsky ◽  
Gonzalo J. Marin ◽  
Iulia Craciun ◽  
Bridget E. Mulvany-Robbins ◽  
...  
Keyword(s):  
Purkinje Cells ◽  
Expression Patterns ◽  
Bird Species ◽  
Glycolytic Enzyme ◽  
Anterior Region ◽  
Zebrin Ii ◽  
Expression Studies ◽  
General Similarity ◽  
Cerebellar Purkinje Cells ◽  
Nothoprocta Perdicaria

Zebrin II (ZII) is a glycolytic enzyme expressed in cerebellar Purkinje cells. In both mammals and birds, ZII is expressed heterogeneously, such that there are sagittal stripes of Purkinje cells with a high ZII expression (ZII+) alternating with stripes of Purkinje cells with little or no expression (ZII-). To date, ZII expression studies are limited to neognathous birds: pigeons (Columbiformes), chickens (Galliformes), and hummingbirds (Trochilidae). These previous studies divided the avian cerebellum into 5 transverse regions based on the pattern of ZII expression. In the lingular region (lobule I) all Purkinje cells are ZII+. In the anterior region (lobules II-V) there are 4 pairs of ZII+/- stripes. In the central region (lobules VI-VIII) all Purkinje cells are ZII+. In the posterior region (lobules VIII-IX) there are 5-7 pairs of ZII+/- stripes. Finally, in the nodular region (lobule X) all Purkinje cells are ZII+. As the pattern of ZII stripes is quite similar in these disparate species, it appears that it is highly conserved. However, it has yet to be studied in paleognathous birds, which split from the neognaths over 100 million years ago. To better understand the evolution of cerebellar compartmentation in birds, we examined ZII immunoreactivity in a paleognath, the Chilean tinamou (Nothoprocta perdicaria). In the tinamou, Purkinje cells expressed ZII heterogeneously such that there were sagittal ZII+ and ZII- stripes of Purkinje cells, and this pattern of expression was largely similar to that observed in neognathous birds. For example, all Purkinje cells in the lingular (lobule I) and nodular (lobule X) regions were ZII+, and there were 4 pairs of ZII+/- stripes in the anterior region (lobules II-V). In contrast to neognaths, however, ZII was expressed in lobules VI-VII as a series of sagittal stripes in the tinamou. Also unlike in neognaths, stripes were absent in lobule IXab, and all Purkinje cells expressed ZII in the tinamou. The differences in ZII expression between the tinamou and neognaths could reflect behavior, but the general similarity of the expression patterns across all bird species suggests that ZII stripes evolved early in the avian phylogenetic tree.

Aldolase C-positive cerebellar Purkinje cells are resistant to delayed death after cerebral trauma and AMPA-mediated excitotoxicity

2007 ◽  
Vol 26 (3) ◽  
pp. 649-656 ◽  
Author(s):  
Jennifer E. Slemmer ◽  
Elize D. Haasdijk ◽  
Doortje C. Engel ◽  
Nikolaus Plesnila ◽  
John T. Weber
Keyword(s):  
Purkinje Cells ◽  
Aldolase C ◽  
Cerebral Trauma ◽  
Delayed Death ◽  
Cerebellar Purkinje Cells

Expression cloning of novel regulators of 92 kDa type IV collagenase expression

2005 ◽  
Vol 33 (5) ◽  
pp. 1135-1136 ◽  
Author(s):  
R.R. Nair ◽  
D.D. Boyd
Keyword(s):  
Dna Sequencing ◽  
Cancer Progression ◽  
Luciferase Reporter ◽  
Expression Cloning ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Type Iv Collagenase ◽  
Expression Library ◽  
Type Iv ◽  
Ht1080 Cells

Overexpression of the 92 kDa type IV collagenase (MMP-9) contributes to cancer progression. However, to date, there are few known regulators of expression of this metalloproteinase. We employed an expression library comprising 500000 cDNA clones to screen for novel regulators of MMP-9 expression. HT1080 cells were transiently co-transfected with an MMP-9 promoter-luciferase reporter and pools of the cDNA expression library. Positive-scoring pools were subdivided in secondary and tertiary screens, after which the regulatory cDNAs were identified by DNA sequencing. This brief review illustrates the utility of expression cloning in identifying specific regulators of MMP-9 expression.

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