Cartilage associated protein (CASP) is a novel developmentally regulated chick embryo protein

1997 ◽  
Vol 110 (12) ◽  
pp. 1351-1359
Author(s):  
P. Castagnola ◽  
M. Gennari ◽  
R. Morello ◽  
L. Tonachini ◽  
O. Marin ◽  
...  
Keyword(s):  
Chick Embryo ◽  
Developmentally Regulated ◽  
Reading Frame ◽  
Amino Terminal ◽  
Nuclear Antigens ◽  
Cartilage Extracellular Matrix ◽  
Low Levels ◽  
Specific Rabbit ◽  
Chicken Protein

A subtracted cDNA library was generated to identify cDNAs specific for chondrocyte mRNAs preferentially expressed at the hypertrophic stage with respect to early differentiation stages. The characterization of a cDNA isolated from this library that hybridizes with a 1.8 kb mRNA is described here. This mRNA is expressed at extremely low levels in dedifferentiated chondrocytes cultured in adherent conditions, at very low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes cultured in suspension conditions. In the developing chick embryo this mRNA is detectable in RNAs extracted from several other tissues besides cartilage. The described cDNA contains a complete open reading frame coding for a polypeptide of about 33 kDa. Homology searches with known cDNA and protein sequences have revealed that the chicken protein is related to the amino-terminal half of two mammalian nuclear antigens. By immunohistochemistry with specific rabbit antisera a strong signal was detected in the cartilage extracellular matrix of selected regions of the developing skeleton. Because of this localization of the antigen we named this protein cartilage associated protein (hereafter referred to as CASP).

Developmental regulation of a novel repetitive protein of Trypanosoma brucei

1987 ◽  
Vol 7 (8) ◽  
pp. 2838-2844
Author(s):  
M R Mowatt ◽  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Signal Sequence ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Membrane Bound

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

scholarly journals Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae

2000 ◽  
Vol 68 (8) ◽  
pp. 4539-4548 ◽  
Author(s):  
M. D. Glew ◽  
L. Papazisi ◽  
F. Poumarat ◽  
D. Bergonier ◽  
R. Rosengarten ◽  
...  
Keyword(s):  
Phase Variation ◽  
Gene Families ◽  
Surface Proteins ◽  
Dna Rearrangements ◽  
Reading Frame ◽  
Mycoplasma Agalactiae ◽  
Amino Terminal ◽  
Clonal Variant ◽  
Variable Surface

ABSTRACT A family of abundant surface proteins (Vpmas [variable proteins ofMycoplasma agalactiae]) undergoing phase variation inM. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An amino-terminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboringvpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes,vpmaX and vpmaY, were orientated divergently and shared highly homologous 5′ untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. ThevpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. ThevpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by thevsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

scholarly journals A 49-Kilodalton Isoform of the Adenovirus Type 5 Early Region 1B 55-Kilodalton Protein Is Sufficient To Support Virus Replication

2009 ◽  
Vol 83 (18) ◽  
pp. 9045-9056 ◽  
Author(s):  
Kathrin Kindsmüller ◽  
Sabrina Schreiner ◽  
Florian Leinenkugel ◽  
Peter Groitl ◽  
Elisabeth Kremmer ◽  
...  
Keyword(s):  
Virus Replication ◽  
Human Tumor ◽  
Adenovirus Type ◽  
Productive Infection ◽  
Virus Growth ◽  
Reading Frame ◽  
Amino Terminal ◽  
Early Region ◽  
Low Levels ◽  
Adenovirus Type 5

ABSTRACT The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48- to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins (E1B-433R) are sufficient for a productive infection.

scholarly journals Developmental regulation of a novel repetitive protein of Trypanosoma brucei.

1987 ◽  
Vol 7 (8) ◽  
pp. 2838-2844 ◽  
Author(s):  
M R Mowatt ◽  
C E Clayton
Keyword(s):  
Trypanosoma Brucei ◽  
Tandem Repeats ◽  
Signal Sequence ◽  
Developmental Regulation ◽  
Developmentally Regulated ◽  
Hydrophobic Domain ◽  
Reading Frame ◽  
Amino Terminal ◽  
Membrane Bound

Trypanosoma brucei undergoes many morphological and biochemical changes during transformation from the bloodstream trypomastigote to the insect procyclic trypomastigote form. We cloned and determined the complete nucleotide sequence of a developmentally regulated cDNA. The corresponding mRNA was abundant in in vitro-cultivated procyclics but absent in bloodstream forms. The trypanosome genome contains eight genes homologous to this cDNA, arranged as four unlinked pairs of tandem repeats. The longest open reading frame of the cDNA predicts a protein of 15 kilodaltons, the central portion of which consists of 29 tandem glutamate-proline dipeptides. The repetitive region is preceded by an amino-terminal signal sequence and followed by a hydrophobic domain that could serve as a membrane anchor; the mRNA was found on membrane-bound polyribosomes. These results suggest that the protein is membrane associated.

scholarly journals Molecular characterization of the Aspergillus nidulans yA locus.

Genetics ◽  
1989 ◽  
Vol 121 (2) ◽  
pp. 249-254 ◽  
Author(s):  
E B O'Hara ◽  
W E Timberlake
Keyword(s):  
Aspergillus Nidulans ◽  
Gene Disruption ◽  
Molecular Organization ◽  
Dna Fragments ◽  
Developmentally Regulated ◽  
Gene Encoding ◽  
Mutant Strains ◽  
Low Levels ◽  
Chromosome I

Abstract We investigated the molecular organization of the region of Aspergillus nidulans chromosome I containing yA, a gene encoding the developmentally regulated enzyme conidial laccase. DNA fragments were identified that complemented the yA2 mutation and were shown to correspond to yA by genetic mapping and gene disruption experiments. The molecular map of the region was oriented to the genetic map by testing DNA fragments for their ability to complement a mutation in the tightly linked adE gene. The yA gene codes for a 2200 nucleotide mRNA that is present at low levels in vegetative cells and mature conidia, but accumulates to high levels in sporulating cultures. yA mRNA appears shortly after differentiation of sporogenous phialide cells. It accumulates in two developmentally abnormal mutant strains that produce phialides but is absent from two mutant strains that do not produce phialides. Thus, yA transcription is probably restricted to phialides. This result is discussed in relationship to the physiological roles played by phialides in spore differentiation.

scholarly journals Cloning, Expression, and Characterization of Aminopeptidase P from the Hyperthermophilic Archaeon Thermococcus sp. Strain NA1

2006 ◽  
Vol 72 (3) ◽  
pp. 1886-1890 ◽  
Author(s):  
Hyun Sook Lee ◽  
Yun Jae Kim ◽  
Seung Seob Bae ◽  
Jeong Ho Jeon ◽  
Jae Kyu Lim ◽  
...  
Keyword(s):  
Metal Binding ◽  
Genomic Analysis ◽  
Enzyme Characterization ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Hyperthermophilic Archaeon ◽  
Amino Terminal ◽  
Aminopeptidase P

ABSTRACT Genomic analysis of a hyperthermophilic archaeon, Thermococcus sp. strain NA1, revealed the presence of a 1,068-bp open reading frame encoding a protein consisting of 356 amino acids with a calculated molecular mass of 39,714 Da (GenBank accession no. DQ144132). Sequence analysis showed that it was similar to the putative aminopeptidase P (APP) of Thermococcus kodakaraensis KOD1. Amino acid residues important for catalytic activity and the metal binding ligands conserved in bacterial, nematode, insect, and mammalian APPs were also conserved in the Thermococcus sp. strain NA1 APP. The archaeal APP, designated TNA1_APP (Thermococcus sp. strain NA1 APP), was cloned and expressed in Escherichia coli. The recombinant enzyme hydrolyzed the amino-terminal Xaa-Pro bond of Lys(N ε-Abz)-Pro-Pro-pNA and the dipeptide Met-Pro (Km , 0.96 mM), revealing its functional identity. Further enzyme characterization showed the enzyme to be a Co2+-, Mn2+-, or Zn2+-dependent metallopeptidase. Optimal APP activity with Met-Pro as the substrate occurred at pH 5 and a temperature of 100°C. The APP was thermostable, with a half-life of >100 min at 80°C. This study represents the first characterization of a hyperthermophilic archaeon APP.

scholarly journals Purification, Gene Cloning, Targeted Knockout, Overexpression, and Biochemical Characterization of the Major Pyrazinamidase fromMycobacterium smegmatis

1998 ◽  
Vol 180 (22) ◽  
pp. 5809-5814 ◽  
Author(s):  
Helena I. M. Boshoff ◽  
Valerie Mizrahi
Keyword(s):  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Overexpression ◽  
Reading Frame ◽  
Amino Terminal ◽  
Dna Library ◽  
Terminal Amino ◽  
Unique Specificity ◽  
Kinetics Of

ABSTRACT The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designatedpzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosispyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparableKm values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaAconferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 μg/ml.

scholarly journals Characterization of a Baculovirus Alkaline Nuclease

2000 ◽  
Vol 74 (14) ◽  
pp. 6401-6407 ◽  
Author(s):  
Lulin Li ◽  
George F. Rohrmann
Keyword(s):  
Essential Gene ◽  
Point Mutations ◽  
Reading Frame ◽  
Baculovirus Gene ◽  
Salt Requirement ◽  
Low Levels ◽  
Alkaline Nuclease ◽  
Maximal Expression ◽  
Rule Out

ABSTRACT All baculovirus genomes sequenced to date encode a homolog of an alkaline nuclease that has been characterized in theHerpesviridae. In this report we describe the characterization of the alkaline nuclease (AN) homolog of theAutographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133). His-tagged AN constructs were expressed in recombinant baculoviruses and affinity purified, and then their enzymatic activity was characterized. AN was found to degrade linear DNA at alkaline pH, preferred Mg2+ over Mn2+, had optimal activity at 35°C, and did not appear to have a salt requirement. To rule out contamination by the endogenous baculovirus gene product or a cellular enzyme, point mutations were introduced into a highly conserved domain of the gene. These mutations were found to markedly reduce or eliminate most of the activity of the affinity-purified enzyme. An antibody generated against the protein was used to analyze its expression by Western blot analysis. AN was found to be expressed at low levels by 12 h postinfection, with maximal expression at 24 h postinfection. Attempts to generate a virus with this gene inactivated were unsuccessful, suggesting that AN may be encoded by an essential gene.

scholarly journals Cloning and Characterization of tdhA, a Locus Encoding a TonB-Dependent Heme Receptor fromHaemophilus ducreyi

1998 ◽  
Vol 66 (9) ◽  
pp. 4254-4262 ◽  
Author(s):  
Christopher E. Thomas ◽  
Bonnie Olsen ◽  
Christopher Elkins
Keyword(s):  
Agar Medium ◽  
Gram Negative Bacteria ◽  
Outer Membrane Receptor ◽  
Gene Encoding ◽  
Reading Frame ◽  
The Family ◽  
Independent Mechanism ◽  
Low Levels

ABSTRACT Haemophilus ducreyi is unable to synthesize heme and must acquire it from its only known host, humans. We cloned and sequenced a gene encoding an outer membrane receptor for heme. It was designated tdhA (for TonB-dependent heme receptor A) since it was related by sequence homology to the family of TonB-dependent receptors. TdhA was strikingly similar to open reading frame HI0113 from the genome of Haemophilus influenzae Rd and also shared homology with five other heme receptors, including HxuC, HemR, HmuR, ChuA, and ShuA, from gram-negative bacteria. AnEscherichia coli hemA tonB mutant strongly expressingH. ducreyi tdhA grew on low levels of heme as a source of heme only when an intact H. ducreyi Ton system plasmid was present, formally demonstrating functional TonB dependence.tdhA was expressed poorly in vitro by H. ducreyi and only under conditions of heme limitation. A survey ofH. ducreyi revealed that all tested strains but one synthesized small amounts of TdhA in vitro under heme-limiting conditions. Surprisingly, an isogenic mutant of tdhA as well as its parent, 35000, both required the same high levels of heme for growth (50 μg/ml [77 μM] on agar medium). This result, together with previous findings, suggests that in vitro, the uptake of heme by H. ducreyi is mediated by a TonB- and TdhA-independent mechanism, possibly diffusion.

Sign in / Sign up

Export Citation Format

Share Document