Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene

A variety of conventional methods allow the expression of multiple foreign proteins in plants by transgene stacking or pyramiding. However, most of these approaches have significant drawbacks. We describe a novel alternative, using a single transgene to coordinate expression of multiple proteins that are encoded as a polyprotein capable of dissociating into component proteins on translation. We demonstrate that this polyprotein system is compatible with the need to target proteins to a variety of subcellular locations, either cotranslationally or posttranslationally. It can also be used to coordinate the expression of selectable marker genes and effect genes or to link genes that are difficult to assay to reporter genes that are easily monitored. The unique features of this polyprotein system are based on the novel activity of the 2A peptide of Foot-and-mouth disease virus (FMDV) that acts cotranslationally to effect a dissociation of the polyprotein while allowing translation to continue. This polyprotein system has many applications both as a research tool and for metabolic engineering and protein factory applications of plant biotechnology.     

The chemokine SDF-1/CXCL12 regulates the migration of melanocyte progenitors in mouse hair follicles

Mouse skin melanocytes originate from the neural crest and subsequently invade the epidermis and migrate into the hair follicles (HF) where they proliferate and differentiate. Here we demonstrate a role for the chemokine SDF-1/CXCL12 and its receptor CXCR4 in regulating the migration and positioning of melanoblasts during HF formation and cycling. CXCR4 expression by melanoblasts was upregulated during the anagen phase of the HF cycle. CXCR4-expressing cells in the HF also expressed the stem cell markers nestin and LEX, the neural crest marker SOX10 and the cell proliferation marker PCNA. SDF-1 was widely expressed along the path taken by migrating CXCR4-expressing cells in the outer root sheath (ORS), suggesting that SDF-1-mediated signaling might be required for the migration of CXCR4 cells. Skin sections from CXCR4-deficient mice, and skin explants treated with the CXCR4 antagonist AMD3100, contained melanoblasts abnormally concentrated in the epidermis, consistent with a defect in their migration. SDF-1 acted as a chemoattractant for FACS-sorted cells isolated from the anagen skin of CXCR4–EGFP transgenic mice in vitro, and AMD3100 inhibited the SDF-1-induced migratory response. Together, these data demonstrate an important role for SDF-1/CXCR4 signaling in directing the migration and positioning of melanoblasts in the HF.     

Mutation spectrum of primary hyperoxaluria type 1 in Tunisia: Implication for diagnosis in North Africa

Background

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive inherited metabolic disease, characterized by progressive kidney failure due to renal deposition of calcium oxalate. Mutations in the AGXT gene, encoding the liver-specific enzyme alanine glyoxylate aminotransferase, are responsible for the disease. We aimed to determine the mutational spectrum causing PH1 and to provide an accurate tool for diagnosis as well as for prenatal diagnosis in the affected families.

Methods

Direct sequencing was used to detect mutations in the AGXT gene in DNA samples from 13 patients belonging to 12 Tunisian families.

Results

Molecular analysis revealed five mutations causing PH1 in Tunisia. The mutations were identified along exons 1, 2, 4, 5 and 7. The most predominant mutations were the Maghrebian “p.I244T” and the Arabic “p.G190R”. Furthermore, three other mutations characteristic of different ethnic groups were found in our study population. These results confirm the mutational heterogeneity related to PH1 in Tunisian population. All the mutations are in a homozygous state, reflecting the high impact of endogamy in our population.

Conclusion

Mutation analysis through DNA sequencing can provide a useful first line investigation for PH1. This identification could provide an accurate tool for prenatal diagnosis, genetic counseling and screen for potential presymptomatic individuals.     

Metabolic reconstructions identify plant 3‐methylglutaconyl‐CoA hydratase that is crucial for branched‐chain amino acid catabolism in mitochondria

The proteinogenic branched‐chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant‐prokaryote comparative genomics detected candidates for 3‐methylglutaconyl‐CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non‐homologous N‐terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein‐fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3‐hydroxymethylglutaryl‐CoA into 3‐methylglutaconyl‐CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark‐induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3‐methylglutaconyl‐CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.     

Genomic structure and characterisation of the promoter region of the human IL-18 gene

Interleukin (IL)-18 is a cytokine that plays an important role in the T helper (Th) 1 response, primarily by its ability to induce gamma interferon (IFN-gamma) production by T cells and Natural Killer (NK) cells. It also plays a role in host resistance to infection and in Lipopolysaccharides (LPS)-induced histopathology. By a direct sequencing on P1 Artificial Chromosomes (PAC) clones, we have determined the genomic structure and the promoter region of the human IL-18 gene. The IL-18 gene spans approximately 20 Kb and consists of 6 exous. The 5'-flanking regions of human IL-18 and mouse IL-18 show 75% homology suggesting conserved promoter regulatory factors.     

Carbon dynamics, development and stress responses in Arabidopsis: involvement of the APL4 subunit of ADP-glucose pyrophosphorylase (starch synthesis)

An Arabidopsis thaliana T-DNA insertional mutant was identified and characterized for enhanced tolerance to the singlet-oxygen-generating herbicide atrazine in comparison to wild-type. This enhanced atrazine tolerance mutant was shown to be affected in the promoter structure and in the regulation of expression of the APL4 isoform of ADP-glucose pyrophosphorylase, a key enzyme of the starch biosynthesis pathway, thus resulting in decrease of APL4 mRNA levels. The impact of this regulatory mutation was confirmed by the analysis of an independent T-DNA insertional mutant also affected in the promoter of the APL4 gene. The resulting tissue-specific modifications of carbon partitioning in plantlets and the effects on plantlet growth and stress tolerance point out to specific and non-redundant roles of APL4 in root carbon dynamics, shoot-root relationships and sink regulations of photosynthesis. Given the effects of exogenous sugar treatments and of endogenous sugar levels on atrazine tolerance in wild-type Arabidopsis plantlets, atrazine tolerance of this apl4 mutant is discussed in terms of perception of carbon status and of investment of sugar allocation in xenobiotic and oxidative stress responses.     

Founder mutations in Tunisia: implications for diagnosis in North Africa and Middle East

ABSTRACT: Background Tunisia is a North African country of 10 million inhabitants. The native background population is Berber. However, throughout its history, Tunisia has been the site of invasions and migratory waves of allogenic populations and ethnic groups such as Phoenicians, Romans, Vandals, Arabs, Ottomans and French. Like neighbouring and Middle Eastern countries, the Tunisian population shows a relatively high rate of consanguinity and endogamy that favor expression of recessive genetic disorders at relatively high rates. Many factors could contribute to the recurrence of monogenic morbid trait expression. Among them, founder mutations that arise in one ancestral individual and diffuse through generations in isolated communities. Method We report here on founder mutations in the Tunisian population by a systematic review of all available data from PubMed, other sources of the scientific literature as well as unpublished data from our research laboratory. Results We identified two different classes of founder mutations. The first includes founder mutations so far reported only among Tunisians that are responsible for 30 genetic diseases. The second group represents founder haplotypes described in 51 inherited conditions that occur among Tunisians and are also shared with other North African and Middle Eastern countries. Several heavily disabilitating diseases are caused by recessive founder mutations. They include, among others, neuromuscular diseases such as congenital muscular dystrophy and spastic paraglegia and also severe genodermatoses such as dystrophic epidermolysis bullosa and xeroderma pigmentosa. Conclusion This report provides informations on founder mutations for 73 genetic diseases either specific to Tunisians or shared by other populations. Taking into account the relatively high number and frequency of genetic diseases in the region and the limited resources, screening for these founder mutations should provide a rapid and cost effective tool for molecular diagnosis. Indeed, our report should help designing appropriate measures for carrier screening, better evaluation of diseases burden and setting up of preventive measures at the regional level.     

Concurrent Bilateral Renal Angiomyolipoma and Renal Cell Carcinoma in a Patient With Tuberous Sclerosis Complex

Renal angiomyolipomas (AMLs) are often associated with tuberous sclerosis. These tumors are predominantly benign, although malignant forms do exist and are known to be associated with renal cell carcinoma. This case report describes a patient with tuberous sclerosis and massive bilateral AML. Total right nephrectomy was performed; histopathologic examination revealed the coexistence of AML and clear cell renal carcinoma in the same kidney. Because differentiation between renal cell carcinoma and AML with minimal or no fat component can be difficult, an accurate diagnosis is critical in the management of renal AML.Key words: Angiomyolipoma, Renal cell carcinoma, Tuberous sclerosisApproximately 80% of patients with tuberous sclerosis complex (TSC) develop renal angiomyolipoma (AML). Although often benign,^1,2 malignant AML and renal cell carcinoma (RCC) have also been reported in patients with TSC. The concurrence of renal AML and RCC in the same kidney has also been reported in patients with TSC and has been revealed on pathologic examination.Several criteria can be used to help predict malignancy in renal AML, such as tumor size, tumor size and necrosis, and atypical mitotic figures. We report a case of associated massive bilateral AML and RCC. We discuss the diagnosis and treatment of renal AML and emphasize the possibility of concurrent renal malignancies in patients with TSC.