Functional characterization of the mouse Serpina1 paralog DOM-7

2018 ◽  
Vol 399 (6) ◽  
pp. 577-582 ◽  
Author(s):  
Karen Jülicher ◽  
Annabell Wähner ◽  
Kerstin Haase ◽  
Karen W. Barbour ◽  
Franklin G. Berger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mouse Models ◽  
Mouse Strain ◽  
Neutrophil Elastase ◽  
Genome Engineering ◽  
Functional Characterization ◽  
Cdna Clones ◽  
High Complexity ◽  
A1at Deficiency

Abstract The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.

scholarly journals Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

1996 ◽  
Vol 7 (10) ◽  
pp. 1535-1546 ◽  
Author(s):  
J P Paccaud ◽  
W Reith ◽  
J L Carpentier ◽  
M Ravazzola ◽  
M Amherdt ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Cdna Clones ◽  
Restrictive Temperature ◽  
Rnase Protection ◽  
Cell Extracts ◽  
Exact Function ◽  
Mammalian Protein

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

scholarly journals Functional characterization of SMN evolution in mouse models of SMA

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erkan Y. Osman ◽  
Madeline R. Bolding ◽  
Eric Villalón ◽  
Kevin A. Kaifer ◽  
Zachary C. Lorson ◽  
...  
Keyword(s):  
Mouse Models ◽  
Functional Characterization

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium

Parasitology ◽  
1998 ◽  
Vol 117 (6) ◽  
pp. 579-588 ◽  
Author(s):  
D. RODRÍGUEZ-CONTRERAS ◽  
P. J. SKELLY ◽  
A. LANDA ◽  
C. B. SHOEMAKER ◽  
J. P. LACLETTE
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Functional Characterization ◽  
Taenia Solium ◽  
Cdna Clones ◽  
Facilitated Diffusion ◽  
Porcine Cysticercosis ◽  
Isolation And Characterization ◽  
Hexose Uptake

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers d-glucose and d-mannose but not by l-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing

2016 ◽  
Vol 75 ◽  
pp. 1-9 ◽  
Author(s):  
Guan-Heng Zhu ◽  
Jun Xu ◽  
Zhen Cui ◽  
Xiao-Tong Dong ◽  
Zhan-Feng Ye ◽  
...  
Keyword(s):  
Genome Editing ◽  
Spodoptera Litura ◽  
Functional Characterization

scholarly journals Structural and functional characterization of elastases from horse neutrophils

1994 ◽  
Vol 300 (2) ◽  
pp. 401-406 ◽  
Author(s):  
A Dubin ◽  
J Potempa ◽  
J Travis
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Proteinase Inhibitors ◽  
Functional Characterization ◽  
Human Neutrophils ◽  
Human Neutrophil Elastase ◽  
Proteinase 3 ◽  
Reactive Site ◽  
Chronic Lung Diseases

In order better to understand the pathophysiology of the equine form of emphysema, two elastinolytic enzymes from horse neutrophils, referred to as proteinases 2A and 2B, have been extensively characterized and compared with the human neutrophil proteinases, proteinase-3 and elastase. Specificity studies using both the oxidized insulin B-chain and synthetic peptides revealed that cleavage of peptide bonds with P1 alanine or valine residues was preferred. Further characterization of the two horse elastases by N-terminal sequence and reactive-site analyses indicated that proteinases 2A and 2B have considerable sequence similarity to each other, to proteinase-3 from human neutrophils (proteinase 2A), to human neutrophil elastase (proteinase 2B) and to a lesser extent to pig pancreatic elastase. Horse and human elastases differed somewhat in their interaction with some natural protein proteinase inhibitors. For example, in contrast with its action on human neutrophil elastase, aprotinin did not inhibit either of the horse proteinases. However, the Val15, alpha-aminobutyric acid-15 (Abu15), alpha-aminovaleric acid-15 (Nva15) and Ala15 reactive-site variants of aprotinin were good inhibitors of proteinase 2B (Ki < 10(-9) M) but only weak inhibitors of proteinase 2A (Ki > 10(-7) M). In summary, despite these differences, the horse neutrophil elastases were found to resemble closely their human counterparts, thus implicating them in the pathological degradation of connective tissue in chronic lung diseases in the equine species.

scholarly journals Sulphonation of dehydroepiandrosterone and neurosteroids: molecular cloning, expression, and functional characterization of a novel zebrafish SULT2 cytosolic sulphotransferase

2003 ◽  
Vol 375 (3) ◽  
pp. 785-791 ◽  
Author(s):  
Takuya SUGAHARA ◽  
Yuh-Shyong YANG ◽  
Chau-Ching LIU ◽  
T. Govind PAI ◽  
Ming-Cheh LIU
Keyword(s):  
Molecular Cloning ◽  
Expressed Sequence Tag ◽  
Expression System ◽  
Functional Characterization ◽  
Amino Acid Identity ◽  
Cdna Clones ◽  
Reverse Transcription Pcr ◽  
Prokaryotic Expression System ◽  
Partial Cdna

By searching the zebrafish EST (expressed-sequence tag) database, we have identified two partial cDNA clones encoding the 5′ and 3′ regions of a putative zebrafish sulphotransferase (ST). Using the reverse transcription-PCR technique, a full-length cDNA encoding this zebrafish ST was successfully cloned. Sequence analysis revealed that this novel zebrafish ST displays 44%, 43% and 40% amino acid identity with mouse SULT2B1, human SULT2B1b and human SULT2A1 ST respectively. This zebrafish ST therefore appears to belong to the SULT2 cytosolic ST gene family. Recombinant zebrafish ST, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as a 34 kDa protein upon SDS/PAGE. Purified zebrafish ST displayed a strong sulphonating activity toward DHEA (dehydroepiandrosterone), with a optimum pH of 9.5. The enzyme also exhibited activities toward several neurosteroids with differential Km and Vmax values. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 °C and 43 °C. Among ten different divalent metal cations tested, Fe2+ and Cd2+ exhibited small, but significant, stimulatory effects, whereas Hg2+ and Cu2+ displayed considerably stronger inhibitory effects on the DHEA-sulphonating activity of the enzyme. These results constitute the first study on the molecular cloning, expression, and characterization of a zebrafish cytosolic SULT2 ST.

scholarly journals Cloning and Functional Characterization of the Early-Lymphocyte-Specific Pb99 Gene

2000 ◽  
Vol 20 (12) ◽  
pp. 4405-4410 ◽  
Author(s):  
Barry P. Sleckman ◽  
Wasif N. Khan ◽  
Wanping Xu ◽  
Craig H. Bassing ◽  
Barbara A. Malynn ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Chromosome 8 ◽  
Cdna Clones ◽  
Reading Frame ◽  
Lymphoid Development ◽  
Membrane Spanning ◽  
G Protein Coupled ◽  
Deficient Mice

ABSTRACT The Pb99 gene is specifically expressed in pre-B cells and thymocytes and not in mature B and T cells or nonlymphoid tissues, implying that it may function in early lymphoid development. We have previously described the cloning of an incomplete cDNA forPb99. Here we report the isolation of full-length cDNAs and genomic clones for the murine Pb99 gene and the mapping of its location to mouse chromosome 8. Sequence analyses of differentPb99 cDNA clones suggest that there may be at least three forms of the Pb99 protein generated by differential processing of the Pb99 transcript. The cDNA with the longest open reading frame encodes a putative protein that has seven hydrophobic domains similar to those of seven membrane-spanning proteins, such as the classical G protein-coupled receptors. To directly address the role of the Pb99 protein in lymphoid development, Pb99-deficient mice were generated by gene targeting, and lymphocyte development in these mice was analyzed.

scholarly journals Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases

2020 ◽  
Vol 48 (7) ◽  
pp. 3722-3733 ◽  
Author(s):  
Eszter Tóth ◽  
Éva Varga ◽  
Péter István Kulcsár ◽  
Virág Kocsis-Jutka ◽  
Sarah Laura Krausz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Plant Cells ◽  
Dependent Manner ◽  
Protospacer Adjacent Motif

Abstract The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.

scholarly journals Characterization and repurposing of the endogenous Type I-F CRISPR–Cas system of Zymomonas mobilis for genome engineering

2019 ◽  
Vol 47 (21) ◽  
pp. 11461-11475 ◽  
Author(s):  
Yanli Zheng ◽  
Jiamei Han ◽  
Baiyang Wang ◽  
Xiaoyun Hu ◽  
Runxia Li ◽  
...  
Keyword(s):  
Genome Editing ◽  
Zymomonas Mobilis ◽  
Genome Engineering ◽  
Functional Characterization ◽  
Type I ◽  
Dna Targeting ◽  
Engineering Practices ◽  
Restriction Modification ◽  
Shed Light

Abstract Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR–Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction–modification (R–M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R–M system. This study might shed light on exploiting and improving CRISPR–Cas systems in other microorganisms for genome editing and metabolic engineering practices.

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