Isolation and characterization of Leishmania mutants defective in glycosomal protein import.

Kinetoplastid parasites contain a unique microbody organelle called the glycosome. Several important metabolic pathways found in the cytoplasm of higher eukaryotes are compartmentalized within the glycosome in these pathogens. This fundamental difference between the host and parasite has led to consideration of the glycosome as a potential chemotherapeutic target. The genetic basis of glycosome biogenesis is therefore of great interest. This report describes the isolation of multiple Leishmania mutant cell lines defective in glycosomal protein import, and the detailed characterization of three such lines. The mutants examined partially mislocalize a subset of glycosomal proteins to the cytosol yet retain wild-type numbers of glycosomes. One of the mutants has a mutation in the previously identified LdPEX2 (GIM1) gene. The other two mutants are demonstrated to contain cell-specific lesions in one or more genes distinct from PEX2. The identification of multiple genetically distinct mutants with defects in glycosome import provides an important genetic tool to facilitate the identification of genes involved in glycosome biogenesis.

A dominant negative mutation in the GIM1 gene of Leishmania donovani is responsible for defects in glycosomal protein localization.

Kinetoplastid protozoa contain a unique microbody organelle called the glycosome. Several important metabolic pathways are compartmentalized within the glycosome that are found in the cytoplasm of higher eukaryotes. We have previously reported the identification of a Leishmania donovani cell line called gim1-1, in which several normally glycosomal proteins are partially mislocalized to the cytoplasm. The GIM1 gene complements the defect and restores import of proteins to the glycosome. Here we demonstrate that GIM1 encodes an integral membrane protein of the glycosome. We also report that the mutant gim1-1 allele behaves as a dominant negative mutation. Introducing the gim1-1 allele extrachromasomally led to mislocalization of a glycosomal reporter protein even in wild-type cells. Gene disruption experiments in heterozygous GIM1/gim1-1 cells showed that when the mutant gim1-1 allele was lost, cells re-established normal glycosomal protein localization. Interestingly, no disruptions of the wild-type allele were obtained. These data indicate that a dominant negative mutation in the GIM1 gene is the sole genetic lesion responsible for the glycosomal defects in gim1-1, and suggest that GIM1 is an essential gene in Leishmania.

Functional identification of a Leishmania gene related to the peroxin 2 gene reveals common ancestry of glycosomes and peroxisomes.

Glycosomes are membrane-bounded microbody organelles that compartmentalize glycolysis as well as other important metabolic processes in trypanosomatids. The compartmentalization of these enzymatic reactions is hypothesized to play a crucial role in parasite physiology. Although the metabolic role of glycosomes differs substantially from that of the peroxisomes that are found in other eukaryotes, similarities in signals targeting proteins to these organelles suggest that glycosomes and peroxisomes may have evolved from a common ancestor. To examine this hypothesis, as well as gain insights into the function of the glycosome, we used a positive genetic selection procedure to isolate the first Leishmania mutant (gim1-1 [glycosome import] mutant) with a defect in the import of glycosomal proteins. The mutant retains glycosomes but mislocalizes a subset glycosomal proteins to the cytoplasm. Unexpectedly, the gim1-1 mutant lacks lipid bodies, suggesting a heretofore unknown role of the glycosome. We used genetic approaches to identify a gene, GIM1, that is able to restore import and lipid bodies. A nonsense mutation was found in one allele of this gene in the mutant line. The predicted Gim1 protein is related the peroxin 2 family of integral membrane proteins, which are required for peroxisome biogenesis. The similarities in sequence and function provide strong support for the common origin model of glycosomes and peroxisomes. The novel phenotype of gim1-1 and distinctive role of Leishmania glycosomes suggest that future studies of this system will provide a new perspective on microbody biogenesis and function.

The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context.

African trypanosomes are extracellular protozoan parasites that are transmitted from one mammalian host to the next by tsetse flies. Bloodstream forms express variant surface glycoprotein (VSG); the tsetse fly (procyclic) forms express instead the procyclic acidic repetitive protein (PARP). PARP mRNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP, VSG and rRNA promoters in permanently-transformed blood-stream and procyclic form trypanosomes, using reporter gene constructs that showed no post-transcriptional regulation. When the constructs were integrated in the rRNA non-transcribed spacer, the ribosomal RNA and VSG promoters were not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid vector sequences was also reduced.

The 3'-untranslated region of membrane exon 2 from the gamma 2a immunoglobulin gene contributes to efficient transcription termination.

Elements of the mouse Immunoglobulin gamma 2a gene, near the membrane-specific poly(A) addition site, were inserted into a heterologous location in either a synthetic mouse gamma 2b gene or a gpt/SV40 chimeric gene and then assayed for their ability to terminate RNA polymerase II transcription in isolated nuclei of transfected myeloma cells. The intact gamma 2a membrane-specific 3'-untranslated region, with its potential stem loop forming sequences and poly(A) site, is able to efficiently terminate transcription in the absence of the downstream region in which transcription normally terminates (term). Termination efficiency in the presence of the termination fragment decreases either when sequences specifying a potential stem/loop, upstream of the poly(A) region, are interrupted or when the stronger membrane poly(A) site is substituted with a weaker, secretory-specific poly(A) site. We therefore conclude that the gamma 2a membrane-specific untranslated region plays a major role in specifying downstream termination. We further conclude that the immunoglobulin gamma 2a, membrane-specific, 3'-untranslated region can function in the context of the gpt gene, driven by an SV40 promoter, to terminate transcription in a poly(A) site dependent fashion.

Use of in vitro-transcribed RNAs as immobilized targets during RNA:RNA hybridization.

We describe the use of in vitro-transcribed complementary RNAs as filter-bound targets during nuclear run-on analyses. Use of these single-stranded reagents in high-stringency RNA:RNA hybridizations increases signal-to-background hybridization seen using DNA targets and allows efficient measurement of transcriptional rates across genes in either direction.

Myelomas and lymphomas expressing the Ig gamma 2a H chain gene have similar transcription termination regions.

During B cell differentiation, the membrane and secretion specific forms of the Ig gamma-H chains of mouse are differentially expressed as a function of the developmental stage of the cell. Representatives of less differentiated and memory B cells (lymphomas) that have undergone the class switch to gamma 2a or gamma 2b H chains produce nearly equal amounts of membrane specific (gamma m) vs secretory specific (gamma s) mRNA. Fully differentiated gamma 2a or gamma 2b plasma cells and their tumors, myelomas, switch to higher levels of gamma s mRNA production relative to gamma m. Selective use of either the gamma s poly(A) site or the downstream gamma m poly(A) site accompanied by specific splicing events could modulate production of these two forms of mature gamma H chain mRNA. Alternatively, transcription termination could be modulated. Through a combination of hybrid protection and in vitro nascent RNA analyses of transcripts from gamma H chain-producing cells arrested at various stages of development, we have mapped transcription termination in both lymphomas (gamma s approximately gamma m mRNA) and in myelomas (gamma s much greater than gamma m) mRNA. Regardless of the developmental stage of the cell, transcription proceeds at a significant level through both the secretory- and membrane-specific poly(A) sites and terminates at least 500 nucleotides downstream of the gamma m poly(A) site in both the gamma 2a and gamma 2b genes. We conclude that transcription termination does not play a major role in the switch to elevated levels of gamma s production in late stage gamma-producing myeloma cells and that alternative RNA processing alone must be responsible for the differential expression of the gamma H chain mRNA.