scholarly journals Molecular cloning of a bovine cathepsin

1985 ◽  
Vol 225 (3) ◽  
pp. 707-712 ◽  
Author(s):  
N J Gay ◽  
J E Walker
Keyword(s):  
Amino Acids ◽  
Cdna Clone ◽  
Untranslated Region ◽  
Maximum Size ◽  
Specific Probe ◽  
Base Pairs ◽  
Hybridization Probe ◽  
Sequence Encoding ◽  
Thiol Proteinases ◽  
Synthetic Oligonucleotides

A cDNA clone for a thiol endoproteinase has been isolated from a bovine heart cDNA library by using a mixture of 32 synthetic oligonucleotides as a hybridization probe. The inserted region is 672 base pairs in length. It contains a sequence encoding the C-terminal region of a protein that is homologous to rat liver cathepsins B and H and to plant thiol proteinases. In addition, it contains the sequence of 442 bases corresponding to the 3′ untranslated region of the mRNA. The inserted region was used as a specific probe in RNA transfer analysis; the size of the mRNA encoding the thiol endoproteinase is estimated to be approx. 1.7 kilobases. Thus, the maximum size of the encoded protein is about 350-400 amino acids.

scholarly journals Molecular cloning of two cysteine proteinases from paw-paw (Carica papaya)

1986 ◽  
Vol 237 (1) ◽  
pp. 105-110 ◽  
Author(s):  
R A McKee ◽  
S Adams ◽  
J A Matthews ◽  
C J Smith ◽  
H Smith
Keyword(s):  
Northern Blot ◽  
Untranslated Region ◽  
Carica Papaya ◽  
Cysteine Proteinase ◽  
Leaf Tissue ◽  
The Other ◽  
Cdna Clones ◽  
Cysteine Proteinases ◽  
Base Pairs ◽  
Hybridization Probe

Two cDNA clones for plant cysteine proteinases have been isolated from a Carica papaya (paw-paw, papaya) leaf tissue cDNA library by using a mixture of 16 synthetic oligodeoxyribonucleotides as a hybridization probe. The inserted regions are 311 and 440 base-pairs in length and have the potential to encode a region corresponding to the C-terminal region of two proteins which are homologous with the known plant cysteine proteinases and the mammalian thiol cathepsins. One of the sequences shows a high (greater than 77%) homology with the plant cysteine proteinase papain, the other is closely related to papaya chymopapain. One sequence contains all, and the other most, of the 3′ untranslated region of the mRNA. The inserts were used as specific probes in Northern Blot analyses giving an estimated size for the two mRNA species of 1.45 kilobases.

Reproducible and variable rearrangements of a Tetrahymena thermophila surface protein gene family occur during macronuclear development

1988 ◽  
Vol 8 (11) ◽  
pp. 5043-5046
Author(s):  
J P Kile ◽  
H D Love ◽  
C A Hubach ◽  
G A Bannon
Keyword(s):  
Environmental Regulation ◽  
Cdna Clone ◽  
Multigene Family ◽  
Protein Gene ◽  
Tetrahymena Thermophila ◽  
Surface Protein ◽  
Surface Proteins ◽  
Hybridization Probe ◽  
Macronuclear Development ◽  
Surface Protein Gene

The expression of Tetrahymena surface proteins serotype H3 (SerH3) and serotype T (SerT) is under environmental regulation. SerH3 is expressed when cells are incubated between the temperatures of 20 and 35 degrees C, while SerT is expressed when cells are grown at temperatures above 35 degrees C. Using a SerH3 cDNA clone as a hybridization probe, we determined that (i) the SerH3 gene is a member of a multigene family; (ii) most members of this multigene family are variably rearranged during macronuclear development; and (iii) the gene which produces the SerH3 mRNA is reproducibly rearranged during macronuclear development.

Molecular cloning and nucleotide sequence analysis of the putative cDNA for the precursor molecule of the chicken LH-β subunit

1989 ◽  
Vol 3 (2) ◽  
pp. 129-137 ◽  
Author(s):  
T. Noce ◽  
H. Ando ◽  
T. Ueda ◽  
K. Kubokawa ◽  
T. Higashinakagawa ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Cdna Clone ◽  
Rabbit Antiserum ◽  
Nucleotide Sequence Analysis ◽  
Cdna Expression Library ◽  
Β Subunit ◽  
Coding Region ◽  
Precursor Molecule ◽  
Cdna Insert ◽  
Hybridization Probe

ABSTRACT A cDNA expression library was constructed from poly(A)+ RNA of broiler chicken adenohypophyses using λ gt11 as a vector. After screening with a rabbit antiserum against chicken LH, a cDNA clone (L12) containing a 436 bp insert was obtained. Using a subclone of L12 in pUC19 (pL12) as the hybridization probe, another cDNA clone (LF127) with a 533 bp insert was isolated. The LF127 contained the full-length cDNA encoding the putative chicken LH-β subunit precursor molecule. Hybridization of the pL12 cDNA insert to adenohypophysial RNA showed that chicken and Japanese quail adenohypophyses contained RNA species of about 0·8 and 1·0 kb respectively. The amount of this RNA species was ten times higher in adult male quails kept under long days at room temperature than in those kept under short days at 7 °C. In-situ hybridization experiments showed the exclusive distribution of the signal in the LH cells of the adenohypophysis. The similarity of the nucleotide sequence of the apoprotein-coding region of LH-β cDNA of the chicken to that of mammals is lower than that among mammals. The deduced amino acid sequence of the chicken LH-β subunit supports the hypothesis that the number of proline residues increases in the LH-β subunit the closer phylogenetically the vertebrate is to mammals.

Identification of two human beta-tubulin isotypes

1983 ◽  
Vol 3 (5) ◽  
pp. 854-862
Author(s):  
J L Hall ◽  
L Dudley ◽  
P R Dobner ◽  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Cdna Clone ◽  
Hela Cells ◽  
Untranslated Region ◽  
Genomic Sequence ◽  
Beta Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Genes ◽  
Processed Pseudogene ◽  
Carboxy Terminal ◽  
High Degree

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.

Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines

1989 ◽  
Vol 9 (5) ◽  
pp. 1832-1838
Author(s):  
K J Kontis ◽  
S M Arfin
Keyword(s):  
Cdna Clone ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mrna Translation ◽  
Resistant Cell ◽  
Trna Synthetase ◽  
Expression Library ◽  
Hybridization Probe ◽  
Ovary Cells

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

Three new components contained in the vitelline coat of Tegula pfeifferi

Zygote ◽  
1999 ◽  
Vol 8 (S1) ◽  
pp. S61-S61 ◽  
Author(s):  
Kazu Haino-Fukushima ◽  
Xuxi Fan ◽  
Shouka Nakamura
Keyword(s):  
Amino Acids ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzymatic Reaction ◽  
Base Pairs ◽  
Sulphated Polysaccharides ◽  
Reading Frame ◽  
Glycosylation Sites ◽  
Long Time ◽  
Protein Components ◽  
Vitelline Coat

The vitelline coat (VC) lysin of Tegula, a marine molluscan genus, is released from the acrosome of sperm during fertilisation and can lyse the VC of only the same species. The lytic action of this lysin against the VC is not an enzymatic reaction, but a stoichiometric and irreversible one (Haino-Fukushima, 1974).The VC of Tegula pfeifferi consists of glycoproteins containing sulphated polysaccharides, which account for roughly two-thirds of the entire weight of the VC. The presence of a large quantity of polysaccharides in the VC had prevented rapid progress in the analysis of its protein components. Last year, we succeeded in a complete solubilisation of the VC by boiling for a long time in 1% SDS solution, and determined the cDNA sequence coding for a mature 41 kDa glycoprotein, which appears to be the major component of the VC from the results of SDS-polyacrylamide gel electrophoresis (PAGE). The cDNA, referred to as vcp41, comprises 1072 base pairs and contains one open reading frame with a sequence for 319 amino acids containing 19 amino acids of a signal peptide. The deduced amino acid sequence has five N-glycosylation sites and ten cysteine residues. It seems that almost 7 kDa in this 41kDa glycoprotein is polysaccharide constituents (Fan & Haino-Fukushima, 1998).

scholarly journals Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 273-283 ◽  
Author(s):  
T Tsujimura ◽  
M Morimoto ◽  
K Hashimoto ◽  
Y Moriyama ◽  
H Kitayama ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mast Cells ◽  
Cell Line ◽  
Point Mutation ◽  
Coding Region ◽  
Juxtamembrane Domain ◽  
Base Pairs ◽  
Constitutive Activation ◽  
Mastocytoma Cells ◽  
Type C

Abstract A peculiar point mutation results in constitutive activation of c-kit receptor tyrosine kinase (KIT) in three different tumor mast cell lines; ie, the HMC-1, P-815, and RBL-2H3. Because constitutive activation of KIT was also observed in the FMA3 mouse mastocytoma cell line, we investigated the molecular mechanism. Sequencing of the whole coding region of the c-kit showed that the point mutation found in HMC- 1, P-815, and RBL-2H3 cells was absent in FMA3 cells and that the c-kit cDNA of FMA3 cells carried an in-frame deletion of 21 base pairs (bp) encoding Thr-Gln-Leu-Pro-Tyr-Asp-His at codons 573 to 579 at the juxtamembrane domain. The FMA3-type c-kit cDNA with 21 bp deletion was introduced into the IC-2 cell line, which was derived from murine cultured mast cells. IC-2 cells were dependent on interleukin (IL)-3 and did not express KIT on the surface. In IC-2 cells introduced with the FMA3-type c-kit cDNA, KIT was constitutively phosphorylated on tyrosines and activated. Moreover, the FMA3-type KIT was dimerized without the stimulation by stem cell factor (SCF), a ligand for KIT. The spontaneously dimerized FMA3-type KIT without SCF binding was not internalized even after the activation. IC-2 cells expressing the FMA3- type KIT grew in suspension culture without IL-3 and SCF and became leukemic in nude athymic mice. The deletion of seven amino acids at the juxtamembrane domain appeared to be a new activating mutation of KIT that might be involved in neoplastic growth of mast cells.

Molecular cloning of the cDNAs encoding pituitary glycoprotein hormone α- and gonadotropin IIβ-subunits of the Japanese eel, Anguilla japonica, and increase in their mRNAs during ovarian development induced by injection of chum salmon pituitary homogenate

1996 ◽  
Vol 16 (2) ◽  
pp. 171-181 ◽  
Author(s):  
M Nagae ◽  
T Todo ◽  
K Gen ◽  
Y Kato ◽  
G Young ◽  
...  
Keyword(s):  
Amino Acids ◽  
Chum Salmon ◽  
Ovarian Development ◽  
Anguilla Japonica ◽  
Japanese Eel ◽  
Base Pairs ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Subunit Mrna ◽  
Pituitary Homogenate

ABSTRACT cDNAs encoding the glycoprotein hormone α- and gonadotropin (GTH) IIβ-subunits of Japanese eel (Anguilla japonica) pituitary were cloned using the polymerase chain reaction. The nucleotide sequence of the glycoprotein hormone α-subunit cDNA was 364 base pairs (bp) long, encoding 117 amino acids, and that of the GTH IIβ-subunit cDNA was 433 bp long, encoding 140 amino acids. The deduced amino acid sequence of each mature subunit showed high homology with those of other teleosts, indicating that the structure of GTH subunits has been conserved during the evolution of teleosts. Changes in the expression of these subunit genes during ovarian development induced artificially by the injection of chum salmon pituitary homogenate were examined using Northern blot analysis. Glycoprotein hormone α-subunit mRNA increased almost linearly during ovarian development, whereas GTH IIβ-subunit mRNA was detected only at the late vitellogenic and migratory nucleus stages. These data indicate that eel GTH II is synthesized mainly at the late vitellogenic and migratory nucleus stages, and suggest that GTH II plays an important role in final oocyte maturation of Japanese eel. Changes in the expression of glycoprotein hormone α- and GTH IIβ-subunits mRNA correlate with the serum estradiol-17β (E2) and testosterone profile during ovarian development. The increase in mRNA of both subunits is probably due to positive feedback of E2 and testosterone produced by ovarian follicles in response to the GTH contained in chum salmon pituitary homogenate.

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