scholarly journals Regulatory elements in the 3′ untranslated region of the GP82 glycoprotein are responsible for its stage-specific expression in Trypanosoma cruzi metacyclic trypomastigotes

Acta Tropica ◽  
2012 ◽  
Vol 123 (3) ◽  
pp. 230-233 ◽  
Author(s):  
Ethel Bayer-Santos ◽  
Luciana Girotto Gentil ◽  
Esteban Maurício Cordero ◽  
Paulo Roberto Ceridório Corrêa ◽  
José Franco da Silveira
Keyword(s):  
Trypanosoma Cruzi ◽  
Untranslated Region ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Metacyclic Trypomastigotes

scholarly journals Trypanosoma cruzi GP63 Proteins Undergo Stage-Specific Differential Posttranslational Modification and Are Important for Host Cell Infection

2009 ◽  
Vol 77 (5) ◽  
pp. 2193-2200 ◽  
Author(s):  
Manjusha M. Kulkarni ◽  
Cheryl L. Olson ◽  
David M. Engman ◽  
Bradford S. McGwire
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Host Cell ◽  
Posttranslational Modification ◽  
Immunofluorescence Microscopy ◽  
Polyclonal Antiserum ◽  
Cell Infection ◽  
Specific Expression ◽  
Metacyclic Trypomastigotes ◽  
Triton X

ABSTRACT The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals In vitro differentiation of Trypanosoma cruzi epimastigotes into metacyclic trypomastigotes using a biphasic medium

2021 ◽  
Vol 2 (3) ◽  
pp. 100703
Author(s):  
Jessica Rodríguez Durán ◽  
Arturo Muñoz-Calderón ◽  
Karina Andrea Gómez ◽  
Mariana Potenza
Keyword(s):  
Trypanosoma Cruzi ◽  
In Vitro Differentiation ◽  
Metacyclic Trypomastigotes ◽  
Biphasic Medium

scholarly journals The transcriptional tissue specificity of the human proα1(I) collagen gene is determined by a negative cis-regulatory element in the promoter

1992 ◽  
Vol 286 (1) ◽  
pp. 179-185 ◽  
Author(s):  
C P Simkevich ◽  
J P Thompson ◽  
H Poppleton ◽  
R Raghow
Keyword(s):  
Tissue Specificity ◽  
Regulatory Element ◽  
Mesenchymal Cell ◽  
Cell Types ◽  
Regulatory Elements ◽  
Negative Influence ◽  
Collagen Gene ◽  
Specific Expression ◽  
Cis Acting ◽  
First Intron

The transcriptional activity of plasmid pCOL-KT, in which human pro alpha 1 (I) collagen gene upstream sequences up to -804 and most of the first intron (+474 to +1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang & Raghow (1991) J. Biol. Chem. 266, 2549-2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-1), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but failed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human pro alpha 1 (I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of pro alpha 1 (I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt -625 to -442 deleted), XbaI (-804 to -331) or SstII (+670 to +1440) resulted in 2-10-fold decreased expression in A204 and HFL-1 cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or XbaI/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the XbaI* deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the XbaI*/(-SstII) construct, which contained the intronic SstII fragment (+670 to +1440) in the reverse orientation, was not expressed in either mesenchymal or nonmesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5′-upstream sequences and the first intron determine the mesenchymal cell specificity of human pro alpha 1 (I) collagen gene transcription.

scholarly journals Fast skeletal muscle-specific expression of a quail troponin I gene in transgenic mice.

1988 ◽  
Vol 8 (12) ◽  
pp. 5072-5079 ◽  
Author(s):  
P L Hallauer ◽  
K E Hastings ◽  
A C Peterson
Keyword(s):  
Skeletal Muscle ◽  
Transgenic Mice ◽  
Troponin I ◽  
Fiber Type ◽  
Regulatory Elements ◽  
Evolutionary Divergence ◽  
Mrna Levels ◽  
Fast Skeletal Muscle ◽  
Specific Expression ◽  
Exon 2

We have produced seven lines of transgenic mice carrying the quail gene encoding the fast skeletal muscle-specific isoform of troponin I (TnIf). The quail DNA included the entire TnIf gene, 530 base pairs of 5'-flanking DNA, and 1.5 kilobase pairs of 3'-flanking DNA. In all seven transgenic lines, normally initiated and processed quail TnIf mRNA was expressed in skeletal muscle, where it accumulated to levels comparable to that in quail muscle. Moreover, in the three lines tested, quail TnIf mRNA levels were manyfold higher in a fast skeletal muscle (gastrocnemius) than in a slow skeletal muscle (soleus). We conclude that the cellular mechanisms directing muscle fiber type-specific TnIf gene expression are mediated by cis-regulatory elements present on the introduced quail DNA fragment and that they control TnIf expression by affecting the accumulation of TnIf mRNA. These elements have been functionally conserved since the evolutionary divergence of birds and mammals, despite the major physiological and morphological differences existing between avian (tonic) and mammalian (twitch) slow muscles. In lines of transgenic mice carrying multiple tandemly repeated copies of the transgene, an aberrant quail TnIf transcript (differing from normal TnIf mRNA upstream of exon 2) also accumulated in certain tissues, particularly lung, brain, spleen, and heart tissues. However, this aberrant transcript was not detected in a transgenic line which carries only a single copy of the quail gene.

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