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.

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.

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.

Prokaryotic expression cloning of a novel human tyrosine kinase

1994 ◽  
Vol 14 (2) ◽  
pp. 982-988
Author(s):  
J F Beeler ◽  
W J LaRochelle ◽  
M Chedid ◽  
S R Tronick ◽  
S A Aaronson
Keyword(s):  
Tyrosine Kinase ◽  
Biochemical Properties ◽  
Serine Phosphorylation ◽  
Kinase Assay ◽  
Expression Cloning ◽  
Cdna Expression Library ◽  
Human Embryonic Lung ◽  
Expression Library ◽  
Cdna Insert

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation

Development ◽  
1993 ◽  
Vol 117 (1) ◽  
pp. 97-104 ◽  
Author(s):  
S.G. Kuhar ◽  
L. Feng ◽  
S. Vidan ◽  
M.E. Ross ◽  
M.E. Hatten ◽  
...  
Keyword(s):  
Granule Cell ◽  
Cerebellar Granule Cell ◽  
Cdna Expression Library ◽  
Cdna Clones ◽  
Cerebellar Granule Neuron ◽  
Expression Library ◽  
Specific Expression ◽  
Cerebellar Granule ◽  
Neuronal Populations ◽  
Changing Patterns

Among CNS neuronal populations, the cerebellar granule cell provides a simple model for analysing the molecular regulation of CNS neurogenesis. In this study, polyclonal antisera raised against immature granule cell precursors, purified from early postnatal mouse cerebellum, were used to isolate 39 unique cDNA clones from a lambda gt11 cDNA expression library made from the same cell population. Northern blot analysis revealed developmental stage and tissue-specific expression of 28 of the clones. In situ localization of mRNAs encoded by these novel cDNAs, as well as those encoding the axonal glycoprotein TAG-1 and the alpha 6 subunit of the GABAA receptor, reveal four distinct stages in cerebellar granule cell differentiation. The developmentally transient and spatially restricted expression of clones GC9 and GC44 identify a previously unrecognized step in cerebellar histogenesis.

scholarly journals Prokaryotic expression cloning of a novel human tyrosine kinase.

1994 ◽  
Vol 14 (2) ◽  
pp. 982-988 ◽  
Author(s):  
J F Beeler ◽  
W J LaRochelle ◽  
M Chedid ◽  
S R Tronick ◽  
S A Aaronson
Keyword(s):  
Tyrosine Kinase ◽  
Biochemical Properties ◽  
Serine Phosphorylation ◽  
Kinase Assay ◽  
Expression Cloning ◽  
Cdna Expression Library ◽  
Human Embryonic Lung ◽  
Expression Library ◽  
Cdna Insert

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

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