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.

PUMA1: a novel protein that associates with the centrosomes, spindle and centromeres in the nematode Parascaris

1998 ◽  
Vol 111 (6) ◽  
pp. 723-735
Author(s):  
M.R. Esteban ◽  
G. Giovinazzo ◽  
A. de la Hera ◽  
C. Goday
Keyword(s):  
Immunofluorescence Microscopy ◽  
Centromeric Region ◽  
Coiled Coil ◽  
Structural Features ◽  
Holocentric Chromosomes ◽  
Expression Library ◽  
Mitotic Apparatus ◽  
Reading Frame ◽  
Spindle Apparatus ◽  
Novel Protein

We have identified a 227 kDa spindle- and centromere-associated protein in Parascaris, designated PUMA1 (Parascaris univalens mitotic apparatus), using a monoclonal antibody (mAb403) generated against Parascaris embryonic extracts. PUMA1 distribution was studied by immunofluorescence microscopy in mitotic and meiotic Parascaris cells, where centromere organization differs greatly. In mitosis, PUMA1 associates throughout cell division with the centrosomes and kinetochore-microtubules, and it concentrates at the continuous centromere region of the holocentric chromosomes. PUMA1 also localizes to the spindle mid-zone region during anaphase and at the midbody during telophase. In meiosis, PUMA1 associates with the centrosomes and with the discrete centromeric regions lacking kinetochore structures. The analysis of colchicine-treated embryos indicated that the association of PUMA1 with the centromeric region depends on microtubule integrity. mAb403 also recognizes spindle components in Drosophila. A series of overlapping cDNAs encoding the gene were isolated from a Parascaris embryonic expression library. Analysis of the nucleotide sequence identified an open reading frame capable of encoding a protein of 227 kDa. Analysis of the protein sequence indicated that PUMA1 is predicted to be a coiled-coil protein containing a large central alpha-helical domain flanked by nonhelical terminal domains. The structural features and cellular distribution of PUMA1 suggest that it may play a role in the organization of the spindle apparatus and in its interaction with the centromere in Parascaris.

scholarly journals Novel TRIM5 Isoforms Expressed by Macaca nemestrina

2007 ◽  
Vol 81 (22) ◽  
pp. 12210-12217 ◽  
Author(s):  
Greg Brennan ◽  
Yury Kozyrev ◽  
Toshiaki Kodama ◽  
Shiu-Lok Hu
Keyword(s):  
Coiled Coil ◽  
Macaca Nemestrina ◽  
Cdna Clones ◽  
Reading Frame ◽  
Spry Domain ◽  
Immunodeficiency Virus ◽  
Hiv Type 1 ◽  
Hiv 1

ABSTRACT The TRIM5 family of proteins contains a RING domain, one or two B boxes, and a coiled-coil domain. The TRIM5α isoform also encodes a C-terminal B30.2(SPRY) domain, differences within which define the breadth and potency of TRIM5α-mediated retroviral restriction. Because Macaca nemestrina animals are susceptible to some human immunodeficiency virus (HIV) isolates, we sought to determine if differences exist in the TRIM5 gene and transcripts of these animals. We identified a two-nucleotide deletion (Δ2) in the transcript at the 5′ terminus of exon 7 in all M. nemestrina TRIM5 cDNA clones examined. This frameshift results in a truncated protein of 300 amino acids lacking the B30.2(SPRY) domain, which we have named TRIM5θ. This deletion is likely due to a single nucleotide polymorphism that alters the 3′ splice site between intron 6 and exon 7. In some clones, a deletion of the entire 27-nucleotide exon 7 (Δexon7) resulted in the restoration of the TRIM5 open reading frame and the generation of another novel isoform, TRIM5η. There are 18 amino acid differences between M. nemestrina TRIM5η and Macaca mulatta TRIM5α, some of which are at or near locations previously shown to affect the breadth and potency of TRIM5α-mediated restriction. Infectivity assays performed on permissive CrFK cells stably transduced with TRIM5η or TRIM5θ show that these isoforms are incapable of restricting either HIV type 1 (HIV-1) or simian immunodeficiency virus infection. The expression of TRIM5 alleles incapable of restricting HIV-1 infection may contribute to the previously reported increased susceptibility of M. nemestrina to HIV-1 infection in vivo.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1435-1442 ◽  
Author(s):  
Edward M. Conway ◽  
Saskia Pollefeyt ◽  
Jan Cornelissen ◽  
Inky DeBaere ◽  
Marta Steiner-Mosonyi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Messenger Rna ◽  
Coiled Coil ◽  
Cdna Clones ◽  
Exon 2 ◽  
Reading Frame ◽  
Coiled Coil Domain ◽  
Acid Protein ◽  
Apoptosis Protein

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murinesurvivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin140, similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin40, lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin140 and survivin121 inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin140 mRNA, whereas survivin121-specific transcripts were detected in all tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis.

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 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.

A member of the HSP90 family from ovineBabesiain China: molecular characterization, phylogenetic analysis and antigenicity

Parasitology ◽  
2015 ◽  
Vol 142 (11) ◽  
pp. 1387-1397 ◽  
Author(s):  
GUIQUAN GUAN ◽  
JUNLONG LIU ◽  
AIHONG LIU ◽  
YOUQUAN LI ◽  
QINGLI NIU ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Amino Acid ◽  
Structural Characteristics ◽  
Heat Shock Protein 90 ◽  
Cellular Transformation ◽  
Amino Acid Sequences ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Reading Frame

SUMMARYHeat shock protein 90 (HSP90) is a key component of the molecular chaperone complex essential for activating many signalling proteins involved in the development and progression of pathogenic cellular transformation. AHsp90gene (BQHsp90) was cloned and characterized fromBabesiasp. BQ1 (Lintan), an ovineBabesiaisolate belonging toBabesia motasi-like group, by screening a cDNA expression library and performing rapid amplification of cDNA ends. The full-length cDNA ofBQHsp90is 2399 bp with an open reading frame of 2154 bp encoding a predicted 83 kDa polypeptide with 717 amino acid residues. It shows significant homology and similar structural characteristics toHsp90of other apicomplex organisms. Phylogenetic analysis, based on the HSP90 amino acid sequences, showed that theBabesiagenus is clearly separated from other apicomplexa genera. Five Chinese ovineBabesiaisolates were divided into 2 phylogenetic clusters, namelyBabesiasp. Xinjiang (previously designated a new species) cluster andB. motasi-like cluster which could be further divided into 2 subclusters (Babesiasp. BQ1 (Lintan)/Babesiasp. Tianzhu andBabesiasp. BQ1 (Ningxian)/Babesiasp. Hebei). Finally, the antigenicity of rBQHSP90 protein from prokaryotic expression was also evaluated using western blot and enzyme-linked immunosorbent assay (ELISA).

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.

A newt type II keratin restricted to normal and regenerating limbs and tails is responsive to retinoic acid

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 497-507 ◽  
Author(s):  
P. Ferretti ◽  
J.P. Brockes ◽  
R. Brown
Keyword(s):  
Retinoic Acid ◽  
Molecular Mechanisms ◽  
Limb Regeneration ◽  
Northern Blotting ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Type Ii ◽  
Expression Library ◽  
Organ Specific ◽  
Wound Epithelium

In order to understand the molecular mechanisms underlying the regenerative ability of the urodele limb, it is important to identify regeneration-associated proteins and to study their regulation. We have recently shown that the anti-cytokeratin monoclonal antibody LP1K reacts strongly with newt blastemal cells, while its reactivity is restricted in normal limbs. By screening a cDNA expression library from the newt blastema with LP1K, we have identified cDNA clones coding for a type II keratin (NvKII) expressed both in the mesenchyme and the specialized wound epithelium of the blastema. While the rod domain of the protein is highly conserved, the homology between NvKII and mammalian type II keratins drops markedly at the N- and C-terminal regions. The expression of this keratin was analysed by Northern blotting and RNAase protection analysis of various newt tissues, and appears to be organ specific, since it is restricted to normal and regenerating limbs and tails. In particular, we have investigated the expression of this keratin mRNA in normal and regenerating limbs. The transcript is barely detectable in the proximal portion of the normal limb, but its level is high in the distal one. After amputation, NvKII mRNA is expressed both in proximal and distal blastemas, although at higher levels distally, indicating that this keratin is regeneration associated. The NvKII transcript is detectable both in mesenchyme and in the wound epithelium of the regenerate, while no transcript is detectable in normal epidermis. The level of NvKII mRNA is markedly down-regulated both in normal and regenerating limbs following intraperitoneal injection with retinoic acid, a putative endogenous morphogen in limb regeneration.

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