Cloning and sequencing of cDNA encoding a rat salivary cysteine proteinase inhibitor inducible by beta-adrenergic agonists

The beta-adrenergic agonist isoproterenol induces a unique secretory protein (LM) in the salivary glands of developing and adult rats. In order to study the regulation of growth and gene expression by catecholamines, we have isolated and sequenced several cDNA clones encoding the LM protein. Each of the LM cDNA clones described identifies, by Northern blot analyses, a single mRNA species of approximately 900 bases in size. The mRNA encoding this secreted protein was not detected in submandibular glands or brains of untreated adult rats. Sequence analyses of the LM cDNA clones revealed a striking similarity to the family 2 of cysteine proteinase inhibitors. Furthermore, when purified LM protein was used to assay for inhibition of cysteine proteinases, the data demonstrated that it is indeed a type of cysteine proteinase inhibitor. This inhibitor, termed rat cystatin S, provides the first example of cysteine proteinase inhibitors that can be induced by beta-adrenergic agonists.     

Phenotypic cost to plants of an extra gene

In lines of transgenic tobacco plants containing cowpea trypsin inhibitor gene constructs, the cost to various phenotypic characteristics has been measured in plants which express the gene at a high level and in plants which possess, but do not express, the cowpea sequences. Small, but in some cases significant, differences between transgenic and untransformed control plants were found in various parameters. There was no additional difference between transgenic plants which expressed cowpea trypsin inhibitor and those which did not. Thus, although the processes of transformation/regeneration may have some small effects on non-targeted phenotypic characteristics, the expression at high levels of this ‘foreign’ protein imposed no additional yield penalty on the plants.     

Identification and characterization of the tumor suppressor p53 in channel catfish (Ictalurus punctatus)

Herein is presented the sequence of a catfish full-length p53 cDNA obtained from a cloned B cell line cDNA library. Southern blot analyses determined that a restriction fragment linked polymorphism (RFLP) existed with PstI among outbred catfish. Western blot analyses demonstrated that, when compared to PBLs, the catfish leukocyte lines express higher levels of p53 protein. Additionally, the results of Western blot analyses and in vitro translation experiments suggest that the catfish leukocyte lines may produce truncated forms of p53 due to internal initiation.     

Serine acetyltransferase involved in cysteine biosynthesis from spinach: molecular cloning, characterization and expression analysis of cDNA encoding a plastidic isoform.

A cDNA clone that encodes a chloroplast-localizing isoform of serine acetyltransferase (SATase) (EC 2.3.1.30) was isolated from spinach (Spinacia oleracea L.). The cDNA encodes a polypeptide of 347 amino acids containing a putative transit peptide of ca. 60-70 amino acids at the N-terminal. Deduced amino acid sequence of SATase from spinach exhibited homology with other SATases from plants. DNA blot hybridization analysis showed the presence of 2-3 copies of Sat gene in the genome of spinach. RNA blot hybridization analysis indicated the constitutive expression of Sat gene in green and etiolated seedlings of spinach. Bacterial expression of the cDNA could directly rescue the cysteine auxotrophy of Escherchia coli caused by a lack of SATase locus (cysE). Catalytically active SATase protein was produced in E. coli cells. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the activity of recombinant spinach SATase, indicating the regulatory function of SATase in this metabolic pathway. A chloroplastic localization of this spinach SATase was revealed by the analyses of transgenic plant expressing transit peptide of SATase-beta-glucuronidase (GUS) fusion protein, and transient expression using the transit peptide-green fluorescent protein (GFP) fusion protein. The result from in vitro translation analysis suggests that this cDNA may encode both plastidic and cytosolic SATases.     

Circadian expression and induction by wounding of tobacco genes for cysteine proteinase

Two sets of clones were isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from tomato cDNA using a degenerate primer based on the sequence of tomato systemin. Contrary to expectation, the clones did not correspond to tobacco homologues of tomato pro-systemin. However, the cDNAs encoded two highly similar proteins with extensive structural homology to cysteine proteinases from a wide range of plant and animal species. Northern blot analyses showed that in unstressed tobacco leaf the genes for the putative proteinases are expressed according to a circadian rhythm. Furthermore, incision wounding enhances the expression approximately six-fold. Other forms of stress, such as infection with tobacco mosaic virus, treatment with ethephon or UV light do not result in induced expression of the tobacco cysteine proteinase genes.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

Cloning and Characterization of Trypsin Inhibitor Gene from Vigna unguiculata

A trypsin inhibitor, member of Bowman-Birk family, was isolated and purified from cowpea (Vigna unguiculata]). Polyclonal antibodies were raised against cowpea trypsin inhibitor (CpTI) protein in rabbits. The gene for CpTI was amplified by polymerase chain reaction and cloned in a bacterial expression vector pVCATFR18416. The expression of CpTI protein in BL21 (DE3) strain of Escherichia coli was confirmed by western blot studies. The CpTI gene was also sequenced and found to exhibit 100% homology with already published sequence of CpTI gene.     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.     

Circadian and senescence-enhanced expression of a tobacco cysteine protease gene

A cDNA clone encoding a cysteine protease was isolated from a tobacco cDNA library, utilizing as a probe a PCR fragment obtained from degenerated primers based on the conserved sequences of plant cysteine protease genes. A putative protein encoded by the clone NTCP-23 had an amino acid sequence with significant similarities to those of plant senescence-associated cysteine proteases and mammalian cathepsin H. Northern blot analysis showed that NTCP-23 mRNA is expressed in all organs and the mRNA and protein expression is enhanced during natural senescence. We propose that NTCP-23 is responsible for amino acid remobilization especially in senescencing leaves. Furthermore, it was found that the mRNA expression follows a circadian rhythm and is reduced by continuous darkness, wounding and hypersensitive reaction (HR). NTCP-23 is the first cysteine protease whose mRNA expression has been shown to be temporarily reduced by wounding.     

A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.     

Molecular cloning and structural and functional characterization of human cathepsin F, a new cysteine proteinase of the papain family with a long propeptide domain.

A cDNA encoding a new cysteine proteinase belonging to the papain family and called cathepsin F has been cloned from a human prostate cDNA library. This cDNA encodes a polypeptide of 484 amino acids, with the same domain organization as other cysteine proteinases, including a hydrophobic signal sequence, a prodomain, and a catalytic region. However, this propeptide domain is unusually long and distinguishes cathepsin F from other proteinases of the papain family. Cathepsin F also shows all structural motifs characteristic of these proteinases, including the essential cysteine residue of the active site. Consistent with these structural features, cathepsin F produced in Escherichia coli as a fusion protein with glutathione S-transferase degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, a substrate commonly used for functional characterization of cysteine proteinases. Furthermore, this proteolytic activity is blocked by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cysteine proteinases. The gene encoding cathepsin F maps to chromosome 11q13, close to that encoding cathepsin W. Cathepsin F is widely expressed in human tissues, suggesting a role in normal protein catabolism. Northern blot analysis also revealed a significant level of expression in some cancer cell lines opening the possibility that this enzyme could be involved in degradative processes occurring during tumor progression.     

Isolation and expression of the full-length cDNA encoding CD59 antigen of human lymphocytes.

To identify the primary structure of CD59 antigen and to elucidate its function, a full-length cDNA clone of CD59 was isolated. The cDNA sequence contained an open reading frame that encodes an 128-amino-acid peptide. The amino-terminal 25 amino acids represented a typical signal peptide sequence and the carboxy-terminal hydrophobic amino acids were characteristic for phosphatidylinositol-anchored proteins. The predicted mature protein sequence showed 35% homology with murine Ly-6C.1 and 31% with Ly-6A.2. The number and the distribution of cysteine residues were conserved, implying that the CD59 represented a human homologue of murine Ly-6. RNA blot hybridization analysis revealed the expression of CD59 mRNA in placental, lung, and pancreatic tissues. The mRNA was not only expressed in T-cell lines but in some of monocytic, myeloid, and B-cell lines. In all of these tissues and cell lines, at least four mRNA species were detected. DNA blot hybridization analysis revealed a rather simple genomic structure, which suggested a single gene as compared with the complex multigene family of murine Ly-6.